Structures of Oxalate Oxidoreductase:
C2 Activation by a Microbial TPP-Dependent
Ferredoxin Oxidoreductase
MASSAcHUSETTS INSTITI ITE
by
OF rEcHNOLOLGY
JUN 24 2015
Marcus Ian Gibson
LIBRARIES
B.S. Chemistry
University of California, Berkeley, 2008
SUBMITTED TO THE DEPARTMENT OF CHEMISTRY IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN INORGANIC CHEMISTRY
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
JUNE 2015
2015 Massachusetts Institute of Technology. All rights reserved.
Signature redacted
Signature of author:
Department of Chemistry
May 5, 2015
Certified by:
Signature redacted
Catherine L. Drennan
Professor of Chemistry and Biology
Howard Hughes Medical Institute Investigator and Professor
Thesis Supervisor
Accepted by:
Signature redacted
I
Robert W. Field
Chairman, Departmental Committee on Graduate Students
1
This doctoral thesis has been examined by a
Committee of the Department of Chemistry as follows:
Signature redacted
11
S1ephen J. Lippard
Committee Chairman
Arthur Amos Noyes Professor of Chemistry
V
Signature redacted
Catherine L. Drennan
Research Supervisor
Professor of Chemistry and Biology
Howard Hughes Medical Institute Investigator and Professor
Signature redacted
Elizabeth M. Nolan
Associate Professor of Chemistry
2
Structures of Oxalate Oxidoreductase:
C2 Activation by a Microbial TPP-Dependent Ferredoxin Oxidoreductase
by
Marcus Ian Gibson
Submitted to the Department of Chemistry
on May 18, 2015 in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy in
Inorganic Chemistry
Abstract
Oxalic acid is a two-carbon diprotic acid that is toxic to humans. In large doses, it can cause
death by poisoning, and in smaller doses over time it can lead to chronic renal disease, such as the
formation of kidney stones, acute renal failure, as well as other complications such as crystalline
arthritis. Current strategies to mitigate oxalate toxicity focus on diet management, though recent
therapeutic studies have begun to focus on both probiotic as well as enzymatic treatments for the
prevention of oxalate-related illnesses.
The acetogenic bacterium Moorella thermoaceticahas been known for some time to metabolize
oxalate, though the unique enzyme responsible was only recently discovered. M thermoacetica
employs an oxalate oxidoreductase (OOR) to oxidize oxalate to two molecules of C0 2 , generating
two low-potential electrons. OOR uses a thiamine pyrophosphate (TPP) cofactor to cleave the C-C
bond, and three [4Fe-4S] clusters to capture and transfer the electrons produced. Both of these
products from oxalate degradation, CO 2 and the low-potential electrons, allow M thermoaceticato
grow via the Wood-Ljungdahl pathway for acetogenesis (also known as the reductive acetyl-CoA
pathway).
OOR is a member of the larger superfamily of 2-oxoacid:ferredoxin oxidoreductase (OFOR)
enzymes. OFORs are found in microorganisms across all domains of life, and are responsible for
performing a number of essential metabolic reactions, including the conversion of pyruvate to
acetyl-CoA. Though, most OFORs require coenzyme A as a co-substrate, OOR is the exception to
this rule, as it is capable of metabolizing oxalate without the aid of CoA.
To aid in understanding this newly discovered enzyme, we have determined three crystal
structures of OOR. These structures have allowed us to visualize the resting state as well as two
reaction intermediates: an oxalate-TPP adduct and a C0 2-TPP adduct. Additionally, these structures
have revealed dramatic protein conformational changes in the active site that are likely to facilitate
catalysis. As OOR is only the second OFOR to be structurally characterized, these structures have
provided a wealth of information about the larger OFOR superfamily as well as about this novel
mechanism of oxalate metabolism.
Thesis Supervisor:
Catherine L. Drennan
Title:
Professor of Chemistry and Biology
Howard Hughes Medical Institute Investigator and Professor
3
Acknowledgments
I would first like to thank Cathy for her support and guidance over the years. I first came to
Cathy because I was interested in metalloenzymes, and she was willing to take me into her group
so that I could learn how to work with proteins and study them. I have gained so much from her,
and I would not have been able to accomplish this work without her guidance. In addition to
her academic mentorship, she has shown exceptional support for me at a personal level, and her
concern for my well-being and for my family members helped me and my family to bear many
unexpected burdens. I would not have been able to finish this degree without her support, both
academically and personally. Cathy also deserves credit for putting together a fantastic research
group. From the time I started until now, the lab has always been a supportive and collaborative
environment that has fostered excellent research.
I would like to thank my thesis committee members, Steve and Liz, for their support over the
years and their specific input and feedback on this thesis. Steve's bioinorganic course in my first
year of graduate school introduced me to the world of metals in enzymes, and I have not looked
back. Steve has always brought valuable insight to our meetings, which has helped me to better
think about the projects I was working on. I have also been grateful for my discussions with
Liz, who likewise has offered good insight into my projects and has always been able to give
suggestions for how to approach specific problems.
Prior to joining the Drennan lab, I got the opportunity to do some work with Jonas Peters,
and though I ended up pursuing a different course in research, my time in his lab gave me a
foundation for thinking about the catalysis of fundamental chemical transformations. I also very
much appreciate the support and mentorship of Jeff Long at Berkeley and his former graduate
student Steven Kaye. Under their mentorship, I began my career in chemical research, a path
which brought me to MIT.
Everything I have done is the result of innumerable conversations, discussions, and consultations
with one or more of my labmates. While it would be true to say that everyone one of my labmates
has helped me at one time or another, a few have done so consistently. Yan, Christine, Christina
anu
I
Le
all helpeU LU
iain me as I sLaieL
a
ITVII. IN LOII
was a
sage,
flielitoR, aid
close Iriend.
Working with Ed has been great. Jeremy is a good friend, and I am glad that we got to share so
much of this MIT experience. I will miss hanging out and discussing science Michael and Marco.
They have helped me immensely in my thinking about enzyme mechanisms. Jen has been a source
of incredible support and encouragement in addition to being a brilliant scientist.
I would not have been able to do the work presented in Chapter 3 without the help of Aileen
Johnson, a talented MIT undergraduate with whom I had the pleasure of working for over two
years. Aileen worked on a number of projects that were quite challenging, and persevered through
many disappointing results. By the end of her time at MIT, she had become proficient in performing
difficult manipulations of crystals in the anaerobic chamber, a skill that enabled us to collect a
dataset that revealed the structure of the oxalate adduct to TPP.
I have been fortunate to have amazing collaborators at the University of Michigan in the lab
of Steve Ragsdale. Steve is always encouraging and every interaction I have had with him has
been both insightful as well as enjoyable. Elizabeth was a good host to me when I visited their lab
during my second year, and I am fortunate that she laid a solid groundwork of biochemistry upon
which to build our understanding of OOR. Mehmet has also been very helpful in making sure we
have enough protein for our experiments, as well as helping us think about OOR activity.
4
My roommates in Tang 1 OB-Sam, Yong, and George-were my constant friends and supports
through the first four years of graduate school, and I am blessed beyond measure to have been
able to know and live with them. As the Intervarsity chaplain, Kevin Ford has been faithful to his
calling for many years, and I am grateful for his shepherding of the Graduate Christian Fellowship,
which has been like family and has taught me to thrive.
For the last three years, I have been blessed with the opportunity to live amongst the smartest
women in the world as a Graduate Resident Tutor in McCormick Hall. The community in
McCormick has had a profound impact on me as I have gotten the opportunity to be a mentor, and
also as I have relied on the friendship and support of our residents and house team. As housemasters,
Charles and Kathy have been models of service and mentorship.
I have been more lucky than most to have Stephanie and Michelle as friends for the last 11
years. It has been a long time since our freshman year at Cal-a lot has happened-and I am so
grateful for their constant support, encouragement, and friendship through moving to the East
Coast and all during our time at MIT. Neither time nor distance shall dissolve this NaCl.
For everything I've done, my parents, Dale and Steve, have done a thousand times more to
support me and help me get to this point. They have always loved me, always encouraged me, and
they have endured much to ensure that I would have the opportunities to do my best. I must also
thank my new parents, Suette and Man-wai, because without their love and support over the last
few years, life would have been much more difficult.
My extended family has also been an enduring source of strength and encouragement. Grandpa
and Grandma especially have always loved me, and I owe so much to them for being living
examples reminding me to keep my eyes set on what is important in life.
My sister Jessi has been a source ofjoy and a constant correction for me. Her faith has put my
own reason to shame, and I am blessed to have her in my life, especially she married such a great
guy as Jon. I am also grateful to have a great brother and sister in Abe and Jenny. I have been glad
for their prayers as they struggle alongside us through Ph.D. life.
I was blessed to meet the most wonderful woman of God in my wife Emily. While I may have
had doubts about coming to MIT initially, I know now that this is where I was meant to be, because
it is here that we met. I could not have asked for a better companion for this latter part of graduate
school, nor a more faithful partner in this world as we struggle daily for the world to come.
The great Scottish athlete Eric Liddell has been a source of inspiration for me, both in my faith
and in my studies. As a Christian, Liddell suffered as a prisoner in China during WWII for the
sake of the Gospel. As a chemist, he wrote an inorganic chemistry textbook from memory in order
to teach the students who were prisoners with him. His reference to Ezekiel in the inscription of
the book, The Bones of Inorganic Chemistry (Can These Dry Bones Live?), was inspirational to
me as I started my journey to understanding the role of metals and other "inorganic" molecules in
biological systems.
To the Father, and to the Son, and to the Holy Spirit, the One God, I give glory for all things.
The One who has brought life out of death has taught me to know joy in all of His Creation, of
which the subject of this work is a part.
5
Thus says the Lord God to these bones: "Surely I will cause breath to enter into you, and you
shall live. I will put sinews on you and bring flesh upon you, cover you with skin and put breath
in you; and you shall live. Then you shall know that I am the Lord."
6
For Mom and Dad,
Grandma and Grandpa
7
8
Contents
Abstract
3
Acknowledgments
4
Abbreviations
Chapter 1
Introduction to oxalate and oxidoreductases
11
13
Summary
13
Acetogenesis and oxalate oxidoreductase
14
Solution chemistry of oxalate
16
Oxalate in biology
18
2-Oxoacid:ferredoxin oxidoreductases
22
References
31
Chapter 2
The structure of oxalate oxidoreductase provides new insight into the
larger family of 2-oxoacid oxidoreductases
37
Summary
37
Contributors
38
Introduction
39
Materials and methods
42
Results
48
Discussion
56
Conclusion
62
Acknowledgments
63
References
63
9
Chapter 3
67
Intermediate-bound structures of OOR reveal enzyme functionality driven
by active-site rearrangement
Summary
67
Contributors
68
Introduction
69
Materials and methods
72
Results
78
Discussion
90
Conclusion
102
Acknowledgments
102
References
103
Chapter 4
107
Oxalate oxidoreductase in context
Summary
107
Introduction
108
Methods
111
Results
116
Discussion
116
Conclusion
120
Acknowledgments
122
References
122
Appendix A - sequence alignments
127
Curriculum Vitae
10
135
Abbreviations
[HE-TPP]
Hydroxyethyl-TPP
ACS
acetyl-CoA synthase
BISDIEN
1,4,10,13,16,22-hexaaza-7,19-dioxacyclotetracosane
CFeSP
corrinoid iron-sulfur protein
CoA
coenzyme A
CODH
carbon monoxide dehydrogenase
D. africanusor Da
Desulfovibrio africanus
DTT
dithiothreitol
IOR
indolepyruvate oxidoreductase
KOR
a-Ketoglutarate (2-oxoglutarate) oxidoreductase
M thermoaceticaor Mt
Moorella thermoacetica
MAD
multiwavelength anomalous dispersion
NCS
non-crystallographic symmetry
0. formigenes
Oxalobacterformigenes
OFOR
2-oxoacid:ferredoxin oxidoreductase
OOR
oxalate oxidoreductase
PDB
Protein Data Bank
PEG
polyethylene glycol
PFOR
pyruvate:ferredoxin oxidoreductase (full-length fusion
protein)
POR
pyruvate:ferredoxin oxidoreductase (composed of multiple
protein chains)
TPP
thiamine pyrophosphate
VOR
ketoisovalerate oxidoreductase
W-L pathway
Wood-Ljungdahl pathway
11
12
Chapter 1
Introduction to oxalate and oxidoreductases
Summary
The work presented in this thesis lies at the intersection of two important fields of biological
study: the metabolism of oxalic acid, and the metabolic conversions performed by anaerobic
2-oxoacid:ferredoxin oxidoreductases (OFORs). Oxalic acid is a molecule that is toxic to humans
and a major cause of renal disease, and yet many organisms have developed ways to break it
down. Of particular interest are microbes that are capable of colonizing the human gut, and
probiotic treatments are currently being investigated to treat renal disease and kidney stones. Five
years ago, our collaborators described a novel enzyme for microbial oxalate metabolism, oxalate
oxidoreductase (OOR). OOR is a member of the OFOR superfamily of enzymes, which is an ancient
and essential family of enzymes found in eukaryotes, bacteria, and archaea. Although a significant
amount of work has been done to study the mechanism of pyruvate:ferredoxin oxidoreductases
(PFORs), which are a related subset of OFORs, prior to this work only one enzyme from the entire
OFOR superfamily had been structurally characterized at high resolution. OOR is the first member
of this superfamily of enzymes that has been found to catalyze the oxidation of oxalate, and the
structures of OOR presented here have shed light not only on the chemistry of this particular family
of enzymes, but on the broader question of how oxalic acid is dealt with in biological systems.
13
Acetogenesis and oxalate oxidoreductase
M thermoaceticafixes carbon dioxide via the Wood-Ljungdahlpathway
Moorella thermoacetica (M thermoacetica, or Mt) (1, 2) is a Gram-positive bacterium that is
the model organism for studying microbial carbon fixation via the reductive acetyl-coenzyme A
(acetyl-CoA) pathway, also known as the Wood-Ljungdahl (W-L) pathway (Figure 1.1) (3). The
W-L pathway allows M thermoacetica,an obligate anaerobe, to grow on carbon dioxide as its sole
source of carbon when there is also a source of low-potential electrons, such as dihydrogen. CO 2 is
reduced by two parallel branches in the W-L pathway, which come together make acetyl-CoA. In the
Eastern or methyl branch, formate dehydrogenase reduces CO 2 to formate, which is subsequently
reduced via a series of folate-dependent steps to the methyl group of methyltetrahydrofolate. This
transformation requires six electrons and seven protons, and produces two molecules of water for
every methyl group formed. From methyltetrahydrofolate, the methyl group is transferred to the
cobalt of the corrinoid iron-sulfur protein (CFeSP), which is able to transfer the methyl group to
the active site of acetyl-CoA synthase (ACS).
In the Western or carbonyl branch, CO 2 is reduced by two electrons and two protons to make
carbon monoxide and water. This reaction takes place at a unique [Ni-3Fe-4S]-Fe cluster called
the C-cluster (4, 5) that is found in the enzyme carbon monoxide dehydrogenase (CODH), which
is part of a complex with ACS. The CO molecule, generated in the CODH subunits, then travels
to the ACS subunits, where it combines with the methyl group from the Eastern branch to make
an acetyl moiety (6). This assembly of the acetyl moiety occurs through an unknown mechanism
at a metallocenter called the A-cluster, in which a labile Ni is linked to a second Ni site as well as
a classical [4Fe-4S] cluster (7, 8). The final step involves nucleophilic attack on the acetyl moiety
by CoA, to form the key cellular metabolite acetyl-CoA.
M thermoaceticacan grow on oxalate via the Wood-Ljungdahlpathway
About 20 years ago it was discovered that, in addition to being used for autotrophic growth
on gas mixtures of CO 2 and H2 , the W-L pathway allowed M thermoacetica to grow on oxalic
acid as its only source of both carbon and energy (9, 10). This pathway was observed to be
14
0
0
oxalate
-
oxi
C )2
C02
2e-, H+
'
0
-0
H
2H, ATP
H20,
tetrahydrofolate
ADP
0
2e- 2H+
N-te trahydrofolate
H
2H+
H 20
NH
carbon monoxide
dehydrogenase
te trahydrofolate
H
2e-
2e-, H+
Nt
H
N
trahydrofolate
'
2e-, H
+
H
H20
H
H -- N-t trahydrofolate
H
corrinoid
H
H-C
,,on-sqffur
protein
CO
H
0
synthase
ATP ADP
-SCoA
P O43-
0-
A OPO 32 - - S oA
acetate
kinase
C02
0
0-crbon
ASCoA
phosphotrar s-
pyruviate-ferredoxin
acetylase
oxidoreductase
Figure 1.1. The Wood-Lungdahl pathway for acetogenesis with oxalate as a carbon and electron source.
Some intermediates have been simplified for clarity.
15
employed even when M thermoaceticawas grown in the presence of nitrate ( l)-a remarkable
observation considering that in most circumstances nitrate is the preferred electron acceptor for
M thermoacetica (12) and that when grown with nitrate, the W-L pathway is usually suppressed
(13, 14). Despite its ability to metabolize oxalate, M thermoacetica is missing the genes for
the oxalyl-CoA decarboxylase pathway (2), which previously was the only known pathway for
anaerobic oxalate metabolism (15). Recently, our collaborators purified and characterized a novel
oxalate oxidoreductase (OOR), which we now understand to be the sole enzyme responsible for
growth on oxalate by M thermoacetica(Figure 1.1) (16).
Solution chemistry of oxalate
Oxalate is unique among C2 molecules. It falls at the interface of organic and inorganic carbon,
with each carbon atom being only one-electron reduced from carbon dioxide. When in its neutralcharge state, oxalic acid is twice protonated as a dicarboxylic acid. However, the pKas of the two
acid groups are quite low, at 1.25 and 3.81 (17) for the first and second proton, respectively, which
means that at physiological pH, oxalate exists as the dianionic conjugate base. In living systems,
therefore, dissolved oxalate consists of two carbons that, although formally in a +3 oxidation state,
are not very electrophilic. This lack of an electrophilic carbon has some important ramifications
when it comes to activating oxalate for metabolism, as we will see. As a hard base that can act as
a bidentate (11 2) ligand, oxalate is capable of forming coordination complexes with many different
metal ions, including Mg 2 ' and Ca2+, salts of which are insoluble (17).
The chemistry of oxalic acid dates back nearly 150 years, when Harcourt et al. described the
decomposition of oxalic acid in a permanganate solution to two molecules of C0 2 , with Mn2+ as
the other product of this reaction (18). As recently as 70 years ago, a mechanism for the anoxic
decomposition of oxalate by Mn3+ was proposed by Duke (19) and Taube (20). The mechanism
consisted of inner-sphere electron transfer, where an oxalate-Mn+ coordination complex was
formed, followed by 1-electron disproportionation and homolytic C-C bond cleavage to form
Mn2+, C0 2 , and a CO 2 '- (Figure 1.2a) (19). The radical CO 2 - species would interact with another
Mn3+ ion, transferring the electron to form a second equivalent of Mn+ and CO 2. It was later
16
Mn 3
2
Mn +
0-
0
0-
2 C02
2 Mn 2
+
0
\
Mn 2
IN
+
a
+
O=C=O
0Mn 3
0
Mn 3+
O/
0-
2002
Mn 2
Mn
+
0
I
b
2
+
Mn 2
+
0
+
0
C
7
0
NH
H
HN
HN ---- Co'+--
~-'C03+---NH
--
Cy-
Fe
-I2.P,/1
O O
HN
0
\.1
0j
0
NH
Cy
""IP
Y
P
Cy
Fe
B-P
,11.1
B-Ph
P
Cy-'
'-Cy
Cy
Cy
H11
Figure 1.2. Oxalate acts as a ligand in coordination compounds. a) Oxalate coordination to Mn3 + was shown
to facilitate cleavage of the C-C bond. In the top pathway, the C-C bond was proposed to undergo homolytic
cleavage following one-electron oxidation, after which a second Mn 3 + ion is required to oxidize the formyl
radical. In the lower pathway, the oxalate radical binds a second Mn3+ ion before undergoing heterolytic
cleavage of the C-C bond to make two molecules of CO2. b) Oxalate and dioxygen are bound in the
(pt-oxalato, ji-peroxo, p-hydroxo)-BISDIEN-dicobalt complex. Oxalate is proposed to undergo homolytic
C-C bond cleavage, followed by inner-sphere electron transfer to the g-peroxo species to form water and
CO 2. c) An Fe(I) complex of the depicted tris(posphino)borate ligand reduces CO2 by one-electron. Two
equivalents of the formyl-Fe(II) species combine to make this g_-i 2 92 -oxalato species.
proposed that the first oxidation step generates a stable oxalate-based radical species that binds a
second Mn3 + atom before the heterolytic C-C bond dissociation occurs with the oxidation of the
radical species (Figure 1.2a) (21). When 02 is present as a terminal electron acceptor, this reaction
requires only catalytic amounts of Mn3" (21).
17
A dicobalt molecule was developed by Martell et al. that makes use of a cryptand ligand to bind
two Co" ions along with bridging peroxide, oxalate, and hydroxide ligands (Figure 1.2b) (22). It was
observed that this compound degraded over time, generating two molecules of C0 2 , two molecules
of water, and an inactive dicobalt(III) species. Inner-sphere electron transfer was invoked for the
homolytic cleavage of the C-C bond of oxalate, with the radical electrons transferring through the
Co3" ions to the p-peroxo ligand.
Recently, Peters et al. synthesized oxalate from CO 2 using an iron(I) catalyst (23). Though
substantially different in nature from the oxalate degradation reaction, the proposed mechanism
of formation of this species invokes a C02'- species bound to the iron coordination complex (24).
This species combines with another molecule of the Fe-CO 2 complex to form the C-C bond and
produce oxalate as a bridging ligand between the two iron atoms (Figure 1.2c). This chemistry, has
also been seen with other elements including lanthanides (25).
Oxalate in biology
Oxalate metabolic pathways
One molecule of oxalate offers very little to organisms that consume it for growth: two carbon
atoms and two reducing equivalents. However, oxalate is a product of the metabolism of many
other biologically important molecules, including vitamin C, and is found in high concentrations
in certain environments. For these reasons, a number of organisms have developed ways to make
use of this otherwise lean molecule. Oxalate metabolism occurs by four known pathways. These
pathways can be separated by the environment in which they operate, either aerobic or anaerobic,
and by the chemistry they perform, either disproportionation or oxidation (Figure 1.3). When
oxalate undergoes disproportionation, the carbon-carbon bond is heterolytically cleaved, generating
CO 2 and formate. When oxalate is oxidized, the carbon-carbon bond is broken, generating two
molecules of C0 2 , with the two electrons reducing a separate electron acceptor.
In aerobic environments, oxalate metabolism is performed by one of two enzymes, both of
which require manganese as a cofactor and are oxygen-dependent, and which activate oxalate
by generating oxalate-based radical species. In oxalate decarboxylases (Figure 1.3a), this radical
18
species undergoes rapid disproportionation, producing CO 2 and formate. In oxalate oxidases
(Figure 1.3b), the oxalate radical initiates C-C bond cleavage, with the reducing equivalents going
to dioxygen to produce hydrogen peroxide.
The enzymes involved in anaerobic oxalate metabolism are dependent on thiamine
pyrophosphate (TPP), a vitamin B, derivative, as a cofactor. TPP is a versatile and ubiquitous
cofactor that is used by organisms across all domains of life as a potent nucleophile capable of
catalyzing many essential biochemical transformations. In the oxalyl-CoA decarboxylase pathway
(Figure 1.3c), CoA first activates oxalate, via a formyl-CoA transferase. The resulting oxalyl-CoA
Aerobic
0
2
+
02, Mn
H+
10
C02
+
+
a
H
oxalate
decarboxylase
0-
Mn 2
+
b
+2 H++02
o
1
2C02
+
H202
oxalate
oxidase
0-
Anaerobic
TPP
+
0
SCoA
-0
0
H
+ 2 Fdox
d
0
0-
0
IN. C02
oxalyl-CoA
decarboxylase
+
C
H
SCoA
TPP
-
2 C02 + 2 Fdred
oxalate
oxidoreductase
Figure 1.3. The four known pathways for oxalate metabolism and their key enzymes. a) Oxalate
decarboxylase catalyzes the disproportionation of oxalate to CO 2 and formate. b) Oxalate oxidase converts
oxalate to 2 molecules of CO 2 and reduces dioxygen to hydrogen peroxide. c) Oxalyl-CoA Decarboxylase
converts oxalyl-CoA to formyl-CoA, generating CO 2 . d) Oxalate oxidoreductase converts oxalate to 2
molecules of CO 2 and reduces 2 equivalents of ferredoxin. a) and b) require molecular oxygen, whereas c)
and d) proceed anaerobically.
19
is a good target for nucleophilic attack by TPP in oxalyl-CoA decarboxylase, which forms an adduct
,
at the thioester carbon of oxalyl-CoA. This adduct undergoes decarboxylation to generate C0 2
followed by protonation and elimination of formyl-CoA. Finally, CoA is reclaimed by formyl-CoA
transferase, releasing formate.
OOR from M thermoaceticarepresents the fourth and final known pathway, and the second
anaerobic pathway, for oxalate metabolism (Figure 1.3d). Like, oxalyl-CoA decarboxylase, OOR
uses TPP. However, TPP in OOR acts directly on the oxalate molecule to form an oxalate-TPP
adduct. This adduct undergoes two distinct decarboxylation steps to generate two molecules of
C0 2, with the electrons captured from the C-C bond being transferred and stored by ferredoxin.
Oxalate in human health
None of these four pathways for oxalate metabolism exist in humans (nor animals in general,
for that matter), and as far as it is known, humans have no mechanism to metabolize oxalate.
This inability to break down oxalate is the root of oxalate toxicity in human health. Oxaluria, the
general term for oxalate-related illness, has various manifestations (26). Simple oxalate poisoning
with high dosages is fatal, and has been recognized since the 19th century, when some of the first
recorded deaths occurred after oxalic acid crystals were mistaken for Epsom salts (26). More
common, however, are the chronic conditions caused by calcium oxalate crystallization within
the body. Kidney stones, which cause intense pain and lead to severe urinary-tract complications,
are primarily composed of calcium oxalate, and in end-stage renal disease, calcium oxalate can
crystalize in the joints, causing a form of arthritis (27).
Kidney stones are a widespread phenomenon, affecting up to 11% of men and 7% of women
in the United States (28). Once formed, stones that are small enough (< 7 mm) are passed through
the urinary tract, an intensely painful process (29). If they are not small enough, they must either be
broken up by extracorporeal shockwave lithotripsy for more easy passage, or surgically removed
(30, 31). Because none of these treatment options are pleasant, stone formers (those who have
developed a kidney stone previously) must make lifestyle changes to try to prevent formation of
20
stones. These changes may mean altering the diet, both to reduce the quantity of oxalate consumed
and to reduce the consumption of oxalate precursors, such as ascorbic acid (vitamin C) (32, 33).
Recently, human microbiome research has addressed the role that microbes play in kidney
stone disease. Colonization of the intestinal tract by the bacterium Oxalobacter formigenes,
which is capable of growth on oxalate, both as an energy and carbon source, has been associated
with a lower risk for stone formation (34, 35). When an 0. formigenes colony is wiped out by
antibiotics (36) or by other circumstances, patients have been found to be more susceptible to stone
formation. This correlation between the make-up of the human gut microbiome and oxalate stone
formation comes from the fact that most oxalate in humans is dietary. When the gut is colonized
with microorganisms that are capable of consuming oxalate, there is less oxalate to be absorbed by
the bloodstream. In addition to probiotic therapies, enzyme therapies that make use of the abovementioned oxalate-degrading enzymes are also being considered for treatment and prevention of
kidney stones (37-40).
Oxalate in plants
Plants have found myriad uses for oxalate, both in solid as well as aqueous phases. Many
plant cells will form razor-sharp calcium oxalate microcrystals that serve as a defense against
predators (41). One example is that of the Tragia ramosa, which is covered in tiny stinging hairs (42).
These hairs are actually individual cells that encase a needle-like calcium oxalate crystal. When
contact is made with the hairs, the cells break and the crystal punctures the skin of the perpetrating
animal, after which a toxin from the plant is channeled down a groove in the needle into the
wound, resulting in skin irritation and itchiness. The crystals themselves can cause irritation, and
are found in high concentrations in a number of plants that are cultivated for food, spinach and
rhubarb being notable examples (43). In addition to defense, these crystals can also function to
regulate calcium availability (41).
Whereas calcium oxalate crystals provide a physical defense, soluble oxalate is also used for
antimicrobial defense in plants. When oxalate is degraded by oxalate oxidases, described above,
21
the product H20 2 can be used to defend against microbial invaders, as evidenced in the response to
powdery mildew fungus by barley roots (44-46).
Oxalate in microorganisms
To microorganisms with the appropriate molecular machinery, oxalate can be a source of both
carbon and energy. Bacillus subtilis expresses one of the few prokaryotic oxalate decarboxylases,
which has a role in metabolism and possibly also in buffering against low-pH environments
(15, 47). The most common prokaryotic oxalate-metabolizing enzyme, however, is oxalyl-CoA
decarboxylase, which may be best known for its role in degrading oxalate in Oxalobacterformigenes
(48-50). 0. formigenes, which was isolated from the human gut, is able to convert oxalate into
CO 2 and formate. It then uses an oxalate:formate antiporter to generate a charge gradient across the
membrane, and so drive the production of ATP. Until the discovery of OOR, these two pathways
were the only known pathways for microbial oxalate metabolism.
2-Oxoacid:ferredoxin oxidoreductases
2-Oxoacid:ferredoxin oxidoreductases (OFORs) are ancient enzymes found across all domains
of life (51), though primarily in strict and facultative anaerobes. As the name implies, these enzymes
perform reversible oxidation/reduction reactions, decarboxylating 2-oxoacids and, with the help of
CoA, forming their respective thioesters. The two electrons captured from the broken C-C bond
are stored in [4Fe-4S] clusters, which can then transfer the electrons to other proteins in the cell.
Thiamine pyrophosphate
The cofactor that performs the chemistry in OFORs is thiamine pyrophosphate (TPP, Figure 1.4).
TPP, which is a derivative of vitamin B 1, or thiamine, is a unique organic catalyst that is capable
of generating a nucleophilic carbon species that then attacks electrophilic substrates (52). TPP is
composed of three parts: an aminopyrimidine group on one end, linked to a methylthiazole ring,
which is further linked to the pyrophosphate moiety on the other end. In enzymes, TPP is bound
primarily through the pyrophosphate group, which coordinates a protein-bound divalent cation
(Mg2 or Ca2' in OFORs). TPP generally binds at the interface of two protein domains. The domain
that binds the pyrophosphate group has a modified Rossmann fold, in which a conserved helix22
helix-strand motif provides residues to coordinate the metal ion, as well as creates a positive dipole
moment that stabilizes the negative pyrophosphate group (Figure 1.5).
The domain binding the aminopyrimidine moiety doesn't play as large a role in binding and
holding TPP, but it is crucial for catalysis. This domain provides a conserved carboxylic acid
sidechain, almost always a glutamate, which forms a strong hydrogen bonding interaction with
Nl' of the pyrimidine ring, stabilizing the iminopyrimidine tautomer (Figure 1.4). Prior to
tautomerization, the lone pair of electrons on the amino nitrogen is involved in the a-bonding
orbitals of the pyrimidine ring, making the amino group a poor nucleophile. However, with the
tautomer stabilized by the protein, the imino-nitrogen lone pair now resides in a non-bonding
(sp2 ) hybrid orbital, which is more nucleophilic than the amino lone pair. This nucleophilic lonepair of electrons is required for deprotonating C2 of the thiazole ring, which activates TPP for
catalysis (52).
The enzyme still faces a challenge in activating TPP, however, because the pKa of the thiazole
C2 is still relatively high, having been determined to be about 17-19 in aqueous solvent (53),
significantly higher than the pKa of the protonated imine. To overcome this barrier, all TPP-
N
H 2N
NH-------- 0
HN
N+
S
%%
0
I
Mg0
0-9
M g2+
S
0
S
0
-% -I
MI+
I
0o90
M g2
+
H
S
/
N+
/
NH-
+H 2N
N+
H
N
0o
1
%0- I
oq O
Mg 2
+
N
N
Figure 1.4. TPP tautomerization and activation of the thiazaole. A conserved glutamate residue in all TPPutilizing enzymes stabilizes the iminopyrimidine tautomer of TPP, allowing deprotonation of C2 of the
thiazole ring.
23
Mg2.
Figure 1.5. The pyrophosphate-binding region of the
The pyrophosphate moiety coordinates a Mg
(residues 110-140 of chain
three Mg
2
+-coordinating
2
1
p chain
in M thermoaceticaoxalate oxidoreductase.
ion in bidentate
(12)
fashion. A helix-helix-strand motif
P, colored red), embedded in the middle of a Rossmann-like domain, provides
residues to hold it in place. Additionally, the N-terminal end of the first helix points
directly at the pyrophosphate moiety, providing its positive dipole to stabilize the negative charges of the
phosphates.
24
0
TPP
A
Tyr
)
lie
C2
Thr
I
Figure 1.6. The active site of Mt OOR demonstrates how the protein facilitates TPP binding in the "V"
conformation. Domain I, or the pyrimidine-binding domain, is colored green whereas domain VI, the
pyrophosphate-binding domain, is colored red. Hydrophobic stacking interactions with TPP rings and
aliphatic carbons are indicated by dashed lines. The substrate-binding pocket to the left of the thiazole ring
is vacant, whereas TPP is stabilized to the right by two Tyr residues. In addition to protein residues, C2 of
the TPP thiazole is labeled.
25
utilizing enzymes prop TPP up in the so-called "V" conformation (Figure 1.6), which places the
imino group of the tautomerized pyrimidine into close proximity of C2. Furthermore, a number
of hydrophobic residues sandwich the TPP rings, providing an environment that is thought to
stabilize the deprotonated thiazole (Figure 1.6) (54).
Whether this activated TPP species is more rightly called a carbanion or a carbene has been
the subject of much discussion over the years (55, 56), though it has been suggested that different
enzymes might stabilize more or less carbene-like species (57). Either way, this deprotonated
thiazole is a potent nucleophile, and is capable of adding itself to electrophilic carbonyls, especially
those that are immediately adjacent to leaving groups (58). When TPP attacks the a-carbonyl
adjacent to a carboxylate, CO 2 is released in a decarboxylation reaction, which leaves an enamine
intermediate. This intermediate can form bonds to carbon, oxygen, nitrogen, and sulfur atoms for
a variety of products.
The resonance capacity of the TPP thiazole is one of the features that allow it to perform
a wide array of difficult reactions. Decarboxylation reactions especially can be difficult as the
resulting negative charge from the disproportionation of a carbon-carbon bond creates a highenergy intermediate. The resonance of TPP allows this negative charge to delocalize over the ring,
stabilizing this intermediate and lowering the barrier to decarboxylation. The intermediate after
CO 2 elimination has been characterized as an "active aldehyde", where the oxo group is nearly coplanar with the thiazole ring, and interacting through a strong hydrogen bond with the sulfur atom
of the thiazole ring (59).
Conversion of metabolites
OFORs perform essential roles in cellular metabolism, allowing cells both to assimilate
molecules from the environment, as well break down metabolites. The best studied of this
superfamily are the pyruvate:ferredoxin oxidoreductases (PFORs). PFORs catalyze the anaerobic
interconversion of pyruvate and acetyl-CoA, both of which are essential cellular metabolites, and
play roles in the biosynthesis of most biomolecules (fatty acids, amino acids, and carbohydrates)
as well as the metabolism of these biomolecules for the production of energy (60).
26
The other classes of OFORs are ketoisovalerate oxidoreductases (VOR), a-ketoglutarate
oxidoreductases (KOR), indolepyruvate oxidoreductases (IOR), and OOR. VORs, KORs, and
IORs are all akin to PFORs in that they require CoA for catalysis, and indeed their product is a
CoA derivative. Whereas PFORs play a central role in microbial metabolism, VORs, KORs, and
IORs are more specialized, being associated primarily with the degradation of branched-chain
aliphatic, carboxylic, and aromatic amino acids, respectively (61-64). Oxalate oxidoreductase, to
our knowledge, is primarily responsible for degrading oxalate to C0 2, which is not a metabolic
intermediate, but can be used by certain autotrophic organisms as a carbon source (3, 16).
Generation of low-potential electrons
In addition to the essential conversion of metabolites, OFORs capture very low-potential
electrons from the C-C bonds that are broken during catalysis. These electrons, with potentials
around -500 mV (see Chapter 4, Table 4.1), are then capable of performing chemistry in a variety of
important and essential cellular processes, ranging from the reduction of protons, carbon dioxide,
and dinitrogen, as well as participating in the degradation of more complex aromatic molecules
(65-68). In Moorella thermoacetica, both PFOR and OOR are capable of providing electrons to
the W-L pathway for the reduction of carbon dioxide the production of acetyl-CoA, and ultimately
pyruvate (Figure 1.1) (16, 69).
The ability of OFORs to capture these low-potential electrons is made possible by [4Fe-4S]
clusters bound to the enzymes. In some enzymes there is only one cluster proximal to the TPP
cofactor (70), which is associated with greater stability to oxygen, whereas in other enzymes, a
separate ferredoxin domain contains two additional clusters that serve as a conduit to the enzyme
surface (71). Iron sulfur clusters are ideal low-potential electron carriers, as they have low
reorganization energies (72). However, they are limited to transferring one reducing equivalent at
a time, necessitating a radical-based mechanism for 2-oxoacid oxidation (73).
The structure andfunction of PFOR
The majority of work that has been performed to understand the biochemistry of OFORs has
been on PFOR. The PFOR from M thermoacetica in particular has been the subject of a number
27
Da PFOR
Figure 1.7. Structure of Da PFOR (PDB ID: 2C42) shown in riboon diagram. The right monomeric unit is
colored gray, while the left monomeric unit is colored by domain as follows: domain I - green; II - blue; III
2
- yellow; IV - orange; V - wheat; VI - red; and VII - pink. TPP is shown as sticks, Mg ' as a green sphere,
and [4Fe-4S] as a ball-and-stick model.
28
of biochemical studies (69, 74), whereas the PFOR from Desulfovibrio africanusis the only PFOR
to be structurally characterized (Figure 1.7) (71, 75, 76). Although the complementarity of these
studies has led to many important breakthroughs in understanding the chemistry of PFOR and
OFORs in general, it has also led to some debate.
Both the PFORs from M thermoacetica (Mt) and D. africanus (Da) are similar in that they
are fusion enzymes, having all of their catalytic domains on one peptide chain. In both instances,
this chain forms a stable dimer that was characterized structurally nearly two decades ago (71).
At the center of the protein is the TPP-binding site. In OFORs, this site is created generally by
two protein subunits, donated a and
P for the pyrimidine-binding
subunit and the pyrophosphate-
binding subunit, respectively. In Da PFOR, the a subunit lies at the N-terminus of the protein chain
corresponding to domains I and II, with domain I binding TPP as mentioned above, and domain II
forming a so-called "transketolase C-terminal domain", which makes dimer interface interactions
in PFOR (77). The P subunit lies at the C-terminus of the protein chain, and corresponds to domain
VI. Domain VI also binds the proximal [4Fe-4S] cluster. In addition to these TPP-binding domains,
OFORs can have a y subunit, domain III in PFOR, which previously had no identified function,
and a 6 subunit, domain V in PFOR, which is the ferredoxin domain, binding the medial and distal
[4Fe-4S] clusters. Domains IV and VII, identified in the structure of Da PFOR, are not conserved
in OFORs, and have no clear function, though domain VII has been associated with increased
oxygen tolerance (78).
The catalytic mechanism of PFOR has been thoroughly studied and debated (Figure 1.8) (73).
One of the spectroscopic signatures of pyruvate oxidation is the formation of a stable hydroxyethylTPP ([HE-TPP]) radical species. This species is generated via a nucleophilic attack by TPP on
pyruvate, followed by decarboxylation and one-electron oxidation. The second one-electron
oxidation step is slow in the absence of CoA, which may be a mechanism the protein uses to guard
against the formation of acetate and loss of this cellular building block. When CoA binds, however,
electron transfer is accelerated by up to three orders of magnitude, and acetyl-CoA is subsequently
eliminated as the reaction product (74).
29
<
C02
*
+I0
_<
0
0S
S
0-
e-
-o
-0
0
CoA
-y
acetyl-CoA
N+
CoAS
e-
S
Figure 1.8. Putative reaction mechanism of pyruvate oxidation by PFOR. A covalent adduct is formed
between TPP and the a-carbon of pyruvate (steps 1 and 2), followed by decarboxylation and formation of a
hydroxyethyl-TPP intermediate (shown deprotonated in step 3). One-electron oxidation of this intermediate
forms a stable radical species (step 4). Attack by CoA forms a tertiary adduct (step 5), which is eliminated
as acetyl-CoA to regenerate the catalyst.
Despite what is known about PFOR chemistry, many of the details of the reaction mechanism,
such as how the protein and coenzyme A in facilitate electron transfer, remain enigmatic.
Additionally, there is still much to be learned about how the other OFORs bind their substrates,
some of them substantially larger than pyruvate, and perform their respective reactions. Many of
these questions require structural information in order to put the biochemical data into its proper
context. However, prior to this work, the structures of Da PFOR were the only high-resolution
structures of an OFOR to be determined. With the structures of OOR presented here, we have been
able to draw conclusions about the mechanism of oxalate oxidation by analogy to that of pyruvate
oxidation - insight not only into a novel pathway for oxalate metabolism, but that also gives us a
new basis for understanding OFOR chemistry.
30
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70. Zhang Q, Iwasaki T, Wakagi T, Oshima T (1996) 2-Oxoacid:Ferredoxin Oxidoreductase from
the Thermoacidophilic Archaeon, Sulfolobus sp. Strain 7. JBiochem (Tokyo) 120(3):587-599.
71. Chabriere E, et al. (1999) Crystal structures of the key anaerobic enzyme pyruvate:ferredoxin
oxidoreductase, free and in complex with pyruvate. Nat Struct Mol Biol 6(2):182-190.
72. Lippard SJ, Berg JM (1994) PrinciplesofBioinorganicChemistry (University Science Books).
73. Reed GH, Ragsdale SW, Mansoorabadi SO (2012) Radical reactions ofthiamin pyrophosphate in
2-oxoacid oxidoreductases. Biochim Biophys Acta BBA - ProteinsProteomics 1824(11):12911298.
74. Furdui C, Ragsdale SW (2002) The Roles of Coenzyme A in the Pyruvate:Ferredoxin
Oxidoreductase Reaction Mechanism: Rate Enhancement of Electron Transfer from a Radical
Intermediate to an Iron-Sulfur Cluster. Biochemistry 41(31):9921-9937.
75. Chabribre E, et al. (2001) Crystal Structure of the Free Radical Intermediate of
Pyruvate:Ferredoxin Oxidoreductase. Science 294(5551):2559-2563.
76. Cavazza C, et al. (2006) Flexibility of Thiamine Diphosphate Revealed by Kinetic
Crystallographic Studies of the Reaction of Pyruvate-Ferredoxin Oxidoreductase with
Pyruvate. Structure 14(2):217-224.
77. Costelloe SJ, Ward JM, Dalby PA (2008) Evolutionary Analysis of the TPP-Dependent Enzyme
Family. JMol Evol 66(1):36-49.
78. Pieulle L, Magro V, Hatchikian EC (1997) Isolation and analysis of the gene encoding the
pyruvate-ferredoxin oxidoreductase of Desulfovibrio africanus, production of the recombinant
enzyme in Escherichia coli, and effect of carboxy-terminal deletions on its stability. JBacteriol
179(18):5684-5692.
35
36
Chapter 2
The structure of oxalate oxidoreductase provides new insight into the larger family of
2-oxoacid oxidoreductases
Summary
Thiamine pyrophosphate (TPP), a derivative of vitamin B1 , is a versatile and ubiquitous
cofactor. When coupled with [4Fe-4S] clusters in microbial 2-oxoacid:ferredoxin oxidoreductases
(OFORs), TPP is involved in catalyzing low-potential redox reactions that are important for the
synthesis of key metabolites and the reduction of N 2, H', and CO 2. We have determined the highresolution (2.27 A) crystal structure of the TPP-dependent oxalate oxidoreductase (OOR), an
enzyme that enables microbes to grow on oxalate, a widely occurring dicarboxylic acid that is found
in soil and freshwater and is responsible for kidney stone disease in humans. OOR catalyzes the
anaerobic oxidation of oxalate, harvesting the low-potential electrons for use in anaerobic reduction
and fixation of CO 2. We compare the OOR structure to the only other structurally characterized
OFOR family member, pyruvate:ferredoxin oxidoreductase. This side-by-side structural analysis
highlights the key similarities and differences that are relevant for the chemistry of this entire class
of TPP-utilizing enzymes.
37
Contributors
Edward J. Brignole (MIT) determined initial crystallization conditions. Elizabeth Pierce
from the Ragsdale lab at the University of Michigan purified OOR from M thermoacetica, and
performed enzyme assays and ensured the quality of the protein. Mehmet Can (UMich) performed
enzyme assays and ensured protein quality. Stephen W. Ragsdale (UMich) and Catherine
L. Drennan provided invaluable guidance and discussion in planning these experiments and
interpreting the results.
38
Introduction
2-Oxoacid:ferredoxin oxidoreductases (OFORs) are an ancient family of enzymes that use
thiamine pyrophosphate (TPP) and three [4Fe-4S] clusters to perform essential carbon-fixation
and redox reactions in microbes. Key to OFOR chemistry is the ability of the TPP cofactor to act as
a potent nucleophile and form covalent adducts with the 2-oxoacid substrates, reversibly cleaving
a carbon-carbon bond and generating electrons capable of reducing low-potential ferredoxins. This
enzyme family predates the divergence of archaea and eukaryotes, and members are ubiquitous
in archaea, common in bacteria, and present in a handful of anaerobic eukaryotes (1, 2). The most
well-studied members of this family are the pyruvate:ferredoxin oxidoreductases (PFORs), which
interconvert pyruvate with acetyl-coenzyme A (acetyl-CoA) and carbon-dioxide (Figure 2.1 a). The
formation of pyruvate from acetyl-CoA and CO 2 by PFOR is required in all modes of anaerobic
CO 2 fixation (3), allowing assimilation of acetyl-CoA into other central metabolites by a number
of different routes: the reductive citric acid cycle in photosynthetic bacteria (4, 5); the WoodLjungdahl (W-L) pathway in acetogenic and sulfate-reducing bacteria as well as in methanogenic
archaea (Figure 2.2) (6); and bi-cycles in several classes of archaea (3). In the opposite direction,
the oxidation of pyruvate by PFOR releases two low-potential electrons (EO' = -515 mV (7, 8);
when pH = 7.0) that can be used by acetogens to reduce CO 2 in the W-L pathway and by many
organisms to reduce dinitrogen to ammonia or protons to hydrogen gas (9-11). Similarly, the
reducing equivalents harvested from a-ketoglutarate oxidation by 2-oxoglutarate:ferredoxin
oxidoreductase can be used by organisms such as Thauera aromatica to reduce and metabolize
0
0-
a.
0
+ -SCoA
SCoA
PFOR
0
o
b.
+ 2e-
C02 +
02CO2
f-(
-o
0
+
2e-
OOR
Figure 2.1. The redox reactions catalyzed by a) PFOR and b) OOR.
39
oxalate
biomass ---
pyruvate
2 H2
OOR
C
+ 6e- +
7H+
CH
,
PFO{R
F
C02
2 e-
The Wood4.jungdahl Pathway
pyruvate
0
FO
ASCoA
Figure 2.2. The overall transformation performed by the Wood-Ljungdahl Pathway in Moorella
thermoacetica and connection to OOR and PFOR. PFOR can operate on both ends of the W-L pathway.
PFOR can cleave pyruvate to contribute both C02 and reducing equivalents to the W-L pathway, and it
can produce pyruvate as a means of assimilating the acetyl-CoA produced by the W-L pathway. In the
presence of oxalate, M. thermoacetica uses OOR to contribute both C02 and reducing equivalents to the
W-L pathway for the production of acetyl-CoA.
aromatic compounds (12). The low-potential of the electrons released by 2-oxoacid oxidation by
OFORs have also allowed for antimicrobial targeting by drugs such as metronidazoles, which
require reductive activation in the potential range of -500 to -260 mV (13).
Despite the ubiquity of this family of enzymes in microbial life and the volume of work that
has been done to understand function, genetics, and evolution, to date only one OFOR has been
structurally characterized to atomic resolution: the PFOR from Desulfovibrioafricanus(DaPFOR)
(14-16). A series of structures of Da PFOR revealed the active site architecture around the TPP
cofactor and allowed for the visualization ofthe arrangement of the three [4Fe 4S] clusters that serve
to transport electrons from the active-site to the protein surface, where they can be transferred to
other redox partners. However, broader applicability of the structure of this enzyme to other OFOR
family members is limited by a handful of peculiar features. Most notably, Da PFOR's domain VII,
which is a 60-residue C-terminal peptide region that interacts directly with the active site and with
the ferredoxin domain, is not found in any other PFORs, nor in any OFOR so far identified. Thus,
the one available view of the active site is unlikely to be representative. Additionally, Da PFOR
40
is part of a subgroup of PFORs that are homodimers of single-chain fusions of the functional
domains, whereas other PFORs are composed of up to four different protein chains in the catalytic
unit (1), suggesting that other differences may be found among this family of enzymes with respect
to subunit-subunit arrangements.
Here we present the second structure of an enzyme in this superfamily, and the first crystal
structure of an oxalate oxidoreductase (OOR). This enzyme from Moorella thermoaceticauses TPP
to oxidize oxalate to two molecules of C0 2 , generating two low-potential electrons (Figure 2. 1b),
which can be transferred to other electron acceptors via three enzyme-bound [4Fe-4S] clusters.
OOR is a member of the OFOR family of enzymes that is unique in that it does not require
CoA, a nucleotide-based organic thiol, for catalysis. It also represents a previously unknown
anaerobic pathway for oxalate metabolism (17-19). Before the discovery of OOR, known
oxalate-metabolizing enzymes fell into one of three metabolic pathways (Figure 1.2). The first
of these pathways is characterized by oxalate oxidases, which produce two molecules of CO 2
while reducing molecular oxygen to H 20 2 , a product that has a role in signaling and defense in
plants (20-22). The second pathway makes use of oxalate decarboxylases, found mostly in fungi
and some bacteria. Oxalate decarboxylases require molecular oxygen for activity, but perform
rather a disproportionation reaction, generating CO 2 and formate from oxalate (23). These first
two pathways share in common a requirement for a manganese cofactor and molecular oxygen for
activity, but do not require CoA. The third pathway, the only pathway previously established to
anaerobically metabolize oxalate, involves oxalyl-CoA decarboxylase, which is a TPP-dependent
enzyme (24). Oxalyl-CoA decarboxylases, like their Mn-dependent counterparts, produce CO 2 and
formate (in the form of formyl-CoA), the latter of which can be used either to generate NADH (25)
or, as in Oxalibacterformgenes,create a membrane potential for the production of ATP (26). As
implied in the name, however, oxalyl-CoA decarboxylases require CoA for activity. In comparison,
OOR is a hybrid of these other oxalate-metabolizing enzymes. It is similar to the aerobic family
members in the lack of requirement for CoA, but the cofactor usage is different (TPP instead of
Mn2') and the oxygen dependence is different, whereas OOR and the other anaerobic oxalate-
41
metabolizing enzyme share the same dependence in their use of one cofactor (TPP), but differ in
use of the other, CoA.
The role of oxalate in biological systems is multi-faceted. As mentioned above, microbes and
plants use electrons generated by its cleavage in processes ranging from energy-generation to
signaling. Humans do not metabolize oxalate and accumulation is associated with kidney stone
formation (27). In the model acetogen, M thermoacetica,the role of oxalate is particularly complex.
At a regulatory level, growth on oxalate up-regulates the W-L pathway, even under conditions in
which the W-L pathway is generally suppressed (28). At a molecular level, cleavage of oxalate
by OOR can provide both the low-potential electrons and the CO 2 substrate for carbon monoxide
dehydrogenase/acetyl-CoA synthase, the key enzyme in this pathway (19). Here our structure of
M thermoacticaOOR provides new insight into the mechanism of this unique pathway for oxalate
metabolism, as well as into the chemistry of the large superfamily of OFORs.
Materials and methods
Protein Purification
OOR was purified from M thermoacetica by the methods described (19), concentrated to
45 mg/ml, as determined by the rose bengal method (29) with a lysozyme standard, and was stored
in a storage buffer of 50 mM Tris, pH 8.0 and 2 mM dithiothreitol at room temperature under an
anoxic nitrogen atmosphere.
Crystallization
OOR was crystallized by hanging drop method at room temperature in a Coy anaerobic chamber
under an Ar/H 2 gas mixture. 30 mg/ml OOR in the storage buffer was mixed with the well solution
(8-11% PEG 3,000, 3-4% Tacsimate, pH 7.0) in a 1:1 ratio to make a 2 pl hanging drop with a
final protein concentration of 15 mg/ml. Crystals large enough for data collection grew in 2-4 days
without any additional crystallization aids. Long, rod-like crystals grew in the orthorhombic space
group P2 12 12 1, but with varying cell constants. The crystal used for collecting multi-wavelength
anomalous dispersion (MAD) data had unit cell constants a = 114 A, b = 145 A, and c = 163 A; the
crystal used for collecting native data had unit cell constants a = 84 A, b = 152 A, and c = 172 A.
42
Before cryocooling, the crystals for MAD and native data were first cryoprotected using a
solution of 15% PEG 3,000, 2% Tacsimate pH 7.0, 20% PEG 400, and 50% storage buffer. Due to
the fragility of the crystals, this cryosolution was added directly to the crystallization drop in two
successive additions of 1 gd and removal of 1 pl from the opposite side of the drop, followed by a
final addition of 2 pl. The drop was allowed to equilibrate for 2 minutes after each addition. After
.
the final addition, crystals were looped and flash-frozen in liquid N 2
Data Collection and Processing
Data were collected at the Advanced Photon Source on Northeastern Collaborative Access
Team beamline 24-ID-C on a Pilatus 6MF detector, making use of a mini kappa goniometer for the
MAD data to ensure simultaneous collection of Bijvoet pairs. HKL 2000 (30) was used to process
all of the datasets. For the MAD datasets, blind boxes were used to block off the top and bottom of
the frame during integration since diffraction was anisotropic and reflections did not extend to the
top and bottom, whereas there were observable reflections extending to the sides. This integration
strategy helped to counter the effects of anisotropy, though it reduced the overall completeness
of the dataset. The native dataset was integrated to 2.27-A resolution, followed by anisotropic
correction using Phaser (31, 32). Data processing statistics are shown in Table 2.1.
Model Building
The structure was determined by iron-multiwavelength anomalous dispersion (MAD)
(Table 2.1). Six iron cluster sites per OOR dimer were found and refined using autoSHARP (33),
with an FOM to 3.51 A resolution of 0.30. The overall phasing power was 0.87. The completeness
of the peak dataset was 77.6%, with the anomalous data good to 5.90-A resolution, where the
phasing power drops below 1.0. Initial electron density maps were obtained after solvent-flattening
to 4.50-A resolution in SHARP (34).
A polyalanine model with [4Fe-4S] clusters (generated with CHAINSAW (32, 35)), based on
D. africanus PFOR (PDB 2C42; a 1.78-A-resolution structure), was placed in the initial electron
density maps using COOT (36). Any parts of this initial model for which there was no electron
density were deleted. Of the six total protein chains in the asymmetric unit, chains A, B, and C
43
Table 2.1. Data Collection and Model Refinement Statistics
MAD
OOR
OOR
Data collection and processing
Space group
Cell dimensions (A)
Dataset
Wavelength (A)
Resolution (A)
Total unique reflections
Completeness (%)
Redundancy
<I/aI>
Rsyml(%)
CCI/2 (%)
P212 12 1
P212 12 1
P212 12 1
P212 12 1
114, 145, 163
114, 145, 163
114, 145, 163
84, 152, 172
Peak*
Inflection
Remote
Native
1.73600
50.0
-
3.50
(3.56 - 3.50)t
50,206
77.6 (44.9)
4.4(4.1)
10.7(2.3)
10.9(54.1)
1.74210
50.0
-
3.80
1.64390
50.0
-
3.75
0.97950
48.7 -2.27
(3.87 - 3.80)
22,509
81.9 (62.4)
(3.81 - 3.75)
24,672
87.1 (82.5)
(2.35 - 2.27)
82,301
81.1 (50.8)
4.8(4.6)
12.2(2.3)
10.9(48.8)
5.0(5.0)
14.4(2.1)
10.1 (54.1)
5.6(4.4)
6.5(2.7)
17.1 (57.7)
(78.8)
Model Refinement
R-work (%)
18.4
R-free (%)
22.2
Protein atoms
15512
TPP-Mg
2
+ atoms
54
[4Fe-4S] atoms
48
Water molecules
945
RMS(bonds) (A)
0.007
RMS(angles) (0)
1.44
Ramachandran favored (%)
97.0
Ramachandran allowed (%)
2.8
Ramachandran outliers (%)
0.2
Average B-factor (A 2 )
Protein (A2)
TPP-Mg2 + (A 2 )
[4Fe-4S] (A 2 )
Water (A 2 )
22.40
22.20
21.82
10.58
26.40
*The peak dataset was scaled anomalously to keep non-equivalent Bijvoet pairs separate. Bijvoet pairs for the
inflection and remote datasets were merged.
Numbers in parenthesis indicate the highest resolution bin.
11(II - <I>1)/Y(I)
The version of HKL2000 used to scale these datasets did not report CCI/2
correspond to one ays unit, and chains D, E, and F correspond to the second ayI3 unit. This initial
model was used as a mask for iterative rounds of density modification in DM (32, 37) (solvent
flattening, histogram matching, 2-fold NCS averaging; the 2-fold non-crystallographic symmetry
44
axis was initially determined with PROFESSS (32)). After a few rounds of building a polyalanine
model, phase combination and extension using the partial model and experimental phases to
3.51-A resolution was performed using SFALL (32, 38), SIGMAA (32, 39), and DM. Side chains
were added and model building continued at 3.51-A resolution with iterative rounds of phase
combination and density modification, until there was negligible improvement in electron density.
Once 88% of the backbone and 46% of sidechains had been built, the partial model was used
as a search model for molecular replacement into the native dataset. Molecular replacement was
performed in Phenix (Phaser-MR) (40) using data to 4.00-A resolution. Phases were quickly
extended, first to 3.00-A resolution, and finally to 2.27-A resolution, allowing the model to be
built to completion through iterative rounds of refinement (simulated annealing and real-space
refinement) using Phenix. NCS restraints were used in early rounds of refinement, but were removed
once most of the sidechains had been added to the model. Geometry restraints for the iron-sulfur
clusters were based on Moorella thermoacetica carbon monoxide dehydrogenase/acetyl-CoA
synthase (PDB 3101) (41), whereas TPP-ligand restraints were based on the crystal structure of
a pyruvate decarboxylase (PDB 2VK8) (42). Distances of cysteine ligands to iron-sulfur clusters
were moderately restrained with a standard deviation of 0.05 A, whereas angles were allowed to
refine freely. The oxidation state of the clusters in our crystals is not known. Distances and angles
of atoms coordinating magnesium were not restrained. Simulated-annealing composite-omit maps
(made with Phenix Autobuild) were used to validate the final model. The model was built to 98.8%
completeness, with the residues in the following chains not having sufficient density to model:
A(l); B(l, 2, 215-219); C(313, 314); D(l, 2); E(, 217-227); F(313, 314). Solvent water molecules
were generated automatically by Phenix using a sigma cutoff of 3.5. Prior to refinement, 5.0% of
unique reflections from the native dataset were set aside as a test dataset, from which Rfrce values
were calculated. Final refinement statistics are shown in Table 2.1.
Accessible surface calculations were performed using Mark Gerstein's program (43), with a
surface probe size of 1.4 A, as implemented at the NIH (44).
45
a.
medial
proximal
TPP
b.
C-terminus
N-terminus
1232 aa
Filii
395 aa
OOR-a
TPP-pyrimidinebinding domain
PFOR
ciD
4iIIcUD
OOR-y
315 aa
Ferredoxin
domain
vi ADOOR-
314 aa
TPP-pyrophosphatebinding domain
Figure 2.3. Structure and domain arrangment of OOR. a) Ribbon drawing of OOR dimer. The right ayp
monomer is colored purple, and the left monomer is colored by domain. The inset highlights the spatial
arrangment of the cofactors of the right monomer. b) Cartoon of domain arrangement for the catalytic units
of Da PFOR on top and OOR (ayp) on the bottom. Domains are indicated with colored boxes, with their
respective domain numbers inside. Black bars connecting domains indicate that the domains are found on
the same polypeptide chain. Ligand-binding function is indicated below the domains of OOR. OOR a forms
46
C.
rjj
Domain
Linker
analog of
Domain IV
111
Mmain
~n
y
Domain 1
IN
domains I (residues 1-250) and II (residues 251-395); OOR y comprises domains III (residues 1-217) and V
(residues 238-315); OOR 0 comprises domain VI (residues 1 314). Domain I binds the pyrimidine moiety
of TPP; domain V binds the medial and distal [4Fe-4S] clusters; domain VI binds the proximal [4Fe-4S]
cluster as well as the pyrophosphate moiety of TPP. Domains IV and VII are only present in Da PFOR. c)
The domains of OOR are shown both individually and in context of the OOR catalytic ayp unit.
47
Results
We solved the structure of M. thermoacetica OOR to 2.27-A resolution in two stages
(Figure 2.3a). First, a low-resolution structure was solved by multi-wavelength anomalous
dispersion methods, and then this low-resolution structure was used for molecular replacement
into a high-resolution dataset with a different unit cell (Table 2.1). The resulting asymmetric unit of
the high resolution structure contains one OOR molecule, which is a dimer of trimers, (0y)
2
(19),
with each ay3 forming a catalytic unit. This oligomeric state is in contrast to Da PFOR, which is a
homodimer, a2 (14), with the catalytic unit composed of a single polypeptide chain. An alignment
of the OOR a, y, and
P chains to the PFOR a chain, however,
shows that both enzymes conserve
the same core domain structure (Figure 2.3b,c) (14, 19). Chain OOR a contains the domains I and
II; chain OOR y, domains III and V; and chain OOR P, domain VI. Notable differences present in
the domain architecture of OOR include the lack of domains IV and VII. The linker that connects
domains III and V in OOR (residues 218-237 of OOR-y) contains no tertiary structure and is thus
not a domain, and the C-terminus of OOR-P occurs before the corresponding start of domain VII
in Da PFOR. Also missing is a four-helix bundle insertion in domain VI of Da PFOR that has
unknown function. Even with these differences, the resemblance between the OOR (ayP) 2 dimer
and the PFOR a2 dimer is striking (Figure 2.4).
The roles of the domains of OOR, when known, also appear to correspond to that in PFOR.
Domain I constitutes one half of the TPP-binding region, providing the residues that interact with
the pyrimidine moiety of thiamine. Domain II is the so-called transketolase C-terminal (TKC)
domain that, apart from being common to the OFOR and transketolase families of enzymes, has
no known function (45). Domain III, unique to the OFORs, likewise has an undetermined function.
Domain V is the ferredoxin domain, coordinating and positioning the medial ([4Fe-4S]med) and
distal ([4Fe-4S]iS) iron-sulfur clusters in the electron transport chain. Domain VI is the second
half of the TPP-binding region, coordinating magnesium and the pyrophosphate moiety of TPP.
Unique among TPP-utilizing enzymes, domain VI in the OFORs also coordinates an iron-sulfur
cluster ([4Fe-4S]prox), which is proximal to the active site.
48
a.
PFOR
b.
OOR
Figure 2.4. Side-by-side comparison of D. africanus PFOR and M thermoaceticaOOR. a) The PFOR Q2
dimer (PDB 2C42) is shown in ribbon representation, with the right monomer colored gray, and the left
monomer colored by domain as follows: I - green, II - blue, III -- yellow, IV - orange, V - wheat, VI -- red,
and VII, pink. b) The GOR (ctyp)2 dimer is shown as in Figure 2.4a.
49
Electron Transfer Chain Is Conserved Between OOR and PFOR
The arrangement of redox cofactors in Da PFOR is almost perfectly conserved in OOR
(Figure 2.5a). Even though domain V is found on a separate chain from domains I and VI, there is
minimal deviation in the positions of the medial and distal clusters, and the distances for electron
transfer differ by no more than 0.4 A. Additionally, conserved protein features around the electrontransfer pathway may give clues as to which features are important for tuning the redox properties
of the clusters, as well as facilitating electron transfer (Figure 2.5b,c). In both OOR and PFOR, the
pyrophosphate moiety of TPP is less than 4 A from Sy of Cys52P (Cys840 in PFOR), which ligates
the proximal cluster. Both enzymes also have Asn143p3 (Asn996 in PFOR), which is poised halfway
a.
FA
TPP
Proxim
1
A4FFe
3
Me
OOR
PFO
C
VI
VI
/"
138
991
G814
G260
H2
N1430
R58y
III
C
A25p
C 8
N996
KS813
K45>II
Figure 2.5. The electron transfer pathway. a) An overlay of the electron transfer pathways of OOR and Da
PFOR (PDB 2C42) shows spatial conservation of the redox cofactors. The structures are aligned by domain
I and VI. OOR is colored as in Figure 2.3, whereas PFOR is gray. [4Fe-4S] clusters are shown in ball-andstick representation, TPP as sticks, and Mg 2* as a green sphere. Domains and cofactors are labeled, as are
nearest-atom distances between the redox-active portions of the cofactors. b) A view of the electron-transfer
pathway in OOR between TPP and the medial [4Fe-4S] cluster. Notable interactions, with interatomic
distances of~4 A or less, are indicated by dashed lines. c) A view of the electron-transfer pathway in PFOR
parallel to that of OOR shown in (b).
50
between the thiazole ring of TPP and the proximal cluster. Also of note is a conserved positivelycharged residue, Arg58y in OOR and Lys459 in PFOR, that lies within hydrogen-bonding distance
of the proximal cluster. Finally, in both OOR and PFOR, the proximal cluster is ligated by a CXGC
(residues 24-27 of OOR-P) motif that also interacts directly with the ferredoxin domain. This motif
is further emphasized by a water molecule, found in both PFOR and OOR structures, positioned
within hydrogen-bonding distance of the glycine backbone-nitrogen, and one of the sulfur atoms
in the medial cluster (Figure 2.5b,c).
One obvious difference in the electron-transfer pathways between OOR and PFOR is the
relative size of the ferredoxin domains (Figure 2.6). A minimal ferredoxin fold with 33% sequence
identity is found in domain V of both enzymes, along with similar electrostatics around the clusters.
PFOR, however, contains a 27-residue insertion that makes its domain V 50% larger than that in
OOR. This insertion does not coordinate the clusters, is primarily solvent-exposed, and its absence
OOR
b.
PFOR
3>r
Figure 2.6. Comparison of ferredoxin domains between OOR and Da PFOR (PDB 2C42). a)Domain V
from OOR is shown in ribbon representation and colored wheat. It is flanked by domains III on the left and
IV on the bottom, colored gray. The medial (left) and distal (right) [4Fe-4S] clusters are shown in ball-andstick representation. b) Domain V from PFOR is shown in ribbon representation and colored wheat. It is
flanked by domains III on the left and IV on the bottom, colored gray. The medial (left) and distal (right)
[4Fe-4S] clusters are shown in ball-and-stick representation. Two insertions above and right of the core
ferredoxin fold create a substantially different protein surface to that in OOR.
51
W
a.
E90a\
D112a
IOOR
E59a
H
O
Y28a
VI
2
115a
R10
TPP
Oo
130a
Q2 l
i
H20
E1100
II
-
2$
F117a
R31 a 'N1380
[4Fe S]
Prox mal
E1 54y"
b.
E 87
I
PFOR
L121
1123
---
A-4E64
H2
3
D963
T31
II i
T991
Met1 202'
[41F
Vill
52
Y28
2VI
pRuvt
prvt
A219'
c
H0
S]
Pro imal
appears to only minimally affect solvent access to the clusters (the distal cluster is 0% and 2.5%
solvent accessible in PFOR and OOR respectively).
SimilarActive Site Structures Provide Clues About Enzyme Specificity
At first glance, the active sites of OOR and PFOR look remarkably similar (Figure 2.7). The
TPP binding motif is functionally conserved, including the strictly-conserved Glu59a (Glu64
in PFOR), as well as a GDGX29YXN (residues 109 143 of OOR P) helix-helix-strand Mg 2+_
pyrophosphate binding motif that is typically found in TPP-utilizing enzymes. Both enzymes also
have a glutamate residue making a potential hydrogen bond with N3' of the TPP pyrimidine ring,
though this residue comes from different domains in each enzyme: Glu90a'* from domain I of the
opposite monomeric unit in OOR and Glu870 from domain VI in PFOR (Figure 2.7, 2.8). Finally,
in OOR Asp 11 2a from domain I interacts indirectly through a water molecule with the imino
group of the TPP pyrimidine, and may have a role in preparing TPP for catalysis, though there is
no analogous interaction in Da PFOR (14).
The similarities between OOR and PFOR also extend to the substrate-binding pocket. For PFOR,
a structure is available depicting substrate (pyruvate)-protein interactions at 1.78-A resolution
(Figure 2.7b). This structure was captured through a crystal-soaking experiment at low pH (6.0)
where the activity of PFOR is substantial diminished (16). Using the position of pyruvate as a
guide, we can compare the active sites of OOR and PFOR (Figure 2.7). We find that both enzymes
*A prime following a residue or domain name (as in Glu9Oc' or domain VII') indicates that a residue or domain is from
the opposite monomeric unit.
Figure 2.7. The active sites of OOR and Da PFOR. a) The active site of OOR is shown with TPP in the
+
middle and the substrate-binding pocket directly to the left, bounded by Argl09a, Gln2 Ia', Phel 17a,
Arg3 Ia, and Asn143P. Some solvent molecules are omitted for clarity. Domains are labeled and colored as
in Figure 2.3. Cofactors and select residue side chains are labeled and shown as sticks. Coordination to Mg2
(green sphere) and important hydrogen-bonding contacts are indicated with dashed lines. b) The active site
of PFOR (PDB 2C42) has TPP, the proximal [4Fe-4S] cluster, and bound pyruvate shown as sticks. The
pyruvate-binding location is analogous to where oxalate binds in OOR. Domains are labeled and colored
as follows: I - green, VI - red, I' and VII' - purple. Coordination to Mg 2+ (green sphere) and important
hydrogen-bonding contacts are indicated with dashed lines.
53
E90a'
0
m2'
OH
0-
,N
I
1
-0
NH
H 20
D112a
E59a
1.
,1 2''
6'
HN
5
7'
m4
+N
-
4
3
5
8 0 A2
6
0p
2
A3
-2
B3
a
N143P
E1103
-O
2bNH
AP -0,
N
S140
0
2
\ BB1 "
-- p -O
/
-O
'
6H 2
O
H2N
B2
N138P
H-bond type
N-H-S
N-H-O
N-H-O
N-H-O
Distance
4.2 A
2.9 A
4.0 A
2.8 A
Potential hydrogen bonding to TPP
TPP atom
Si
H-bond partner residue
N143p
NI'
NY
Nam4'
E59a
E90a'
H2 0
Figure 2.8. Numbering scheme in TPP and its immediate environment. Thiamine pyrophosphate is shown
in the ylide form with C2 deprotonated, with atoms numbered for reference. The mode of binding of the
pyrophosphates to Mg 2l, as well as the rest of the Mg 2+ coordination sphere, is shown. Potential hydrogenbonding interactions between the thiazole and pyrimidine rings of TPP and the surrounding environment
are indicated by dashed lines. Protonation states are hypothesized. Distances between residues in the OOR
crystal structure are indicated in the table.
54
have Arg 1 09a (Arg 114 in PFOR) directed towards the active site; this arginine can hydrogenbond with pyruvate in PFOR and provide a balancing positive charge to the negatively-charged
pyruvate, a functionality that could carry over to oxalate binding in OOR. Asn143P, mentioned
above with respect to the electron-transfer pathway, is also positioned to interact with substrate in
the active site.
One substantial difference in the substrate-binding pocket was noted by sequence
alignments (19). A YPITP active-site motif that is conserved in PFORs (residues 28-32 in Da
PFOR) is altered to a YPIRP motif in OOR (residues 28-32 of OOR-a). In this motif, Tyr and Ile
help to prop up TPP in the so-called "V-conformation" that facilitates catalysis (Figure 2.7b) (46).
In PFOR, Thr of the YPITP motif provides hydrogen-bonding interactions to bound pyruvate. The
substitution of Thr to Arg in OOR was hypothesized to provide an extra positively-charged residue
in the substrate-binding pocket, to facilitate binding of the additionally negatively charged oxalate
molecule (19). The structure of OOR reveals that indeed, Arg31 a is positioned to do just that.
Arg3 Ica also substitutes for Met1202' of Da PFOR in providing a floor to the substrate-binding
pocket (Figure 2.7). Interestingly, Metl202' is from domain VII' of Da PFOR, and is not present
in other PFORs or OFORs. In Da PFOR this domain extends from one monomer to wrap around
and insert into the active site of the other monomer (Figure 2.9). OOR fills part of this void with
Arg3Ia (Figure 2.7).
Three other active-site substitutions in OOR are (PFOR-+OOR): Leul2l--Glyl15a,
Ile123-*Phe1 17a, and Ala219'-+Gln2l a'. The first two substitutions remove large aliphatic sidechains from the active site, which in PFOR serve to provide hydrophobic interactions with the
methyl group of pyruvate. Opening up the OOR active site in this way makes room for the third
substituted residue, Gln2 11a' from domain I of the opposite monomer, to provide for hydrophilic
interactions in the substrate-binding pocket. Taken together, these active-site substitutions in OOR
make the substrate-binding pocket substantially more hydrophilic and potentially more amenable
to oxalate binding than the active site of PFOR.
55
Lack of a Domain VII Does Not Necessarily Create a More Accessible Active Site
In all structures of the Da PFOR enzyme (14-16), access to the active site is blocked by domain
VII of the adjacent monomer (Figure 2.9), raising the question of how a large substrate like CoA
is able to reach the TPP cofactor. OOR does not have domain VII and we were expecting to find a
more open active site as a result. However, we find that a 13-residue insert into domain III-one
of the few large inserts in OOR relative to PFOR-transforms what is a small turn in PFOR into
an extended loop that occupies the same cavity in OOR as domain VII in PFOR (Figure 2.10). In
addition to restricting solvent-access to the active site, this domain III loop has a glutamate residue
(Glul54y) that forms a salt-bridge to Arg31a (the residue that replaces PFOR's Met1202' from
domain VII'). Thus, contrary to our expectations, solvent access to TPP is not as different in OOR
from that in PFOR.
Discussion
OOR is only the second enzyme of the superfamily of 2-oxoacid:ferredoxin oxidoreductases
to be structurally characterized. The similarities between OOR and Da PFOR are reflected in
the domain architecture, which is conserved within the OFOR family of enzymes. Apart from
the requirement for TPP, the similarities between these enzymes are best understood from the
perspective of the low-potential electrons in the substrate carbon-carbon bond.
In PFOR, pyruvate is first decarboxylated to form a TPP-hydroxyethyl [HE-TPP] species,
which then undergoes two one-electron oxidations involving a chain of three [4Fe-4S] clusters
(47, 48). The crystal structure of Da PFOR revealed the spatial arrangement of the three [4Fe-4S]
clusters, which are coordinated by domains V and VI (14). The distances between all the redox
cofactors-TPP to [4Fe-4S]Ox to [4Fe-4S]med to [4Fe-4S]diS,-are all within the range for electron
transfer between protein-bound redox-cofactors. What is more, the medial and distal clusters are
positioned close to the surface of the protein, where they can transfer electrons to other electrontransfer proteins or directly to other redox enzymes.
Here we find that the three [4Fe-4S] clusters of OOR are positioned almost identically despite
substantial deletions in domains V and VI of OOR relative to Da PFOR. Domain V especially,
56
b.
Figure 2.9. Domain VII of Da PFOR (PDB-ID: 2C42). a) The PFOR a 2 dimer is shown in surface
representation. The left monomer is colored gray. The right monomer is shown with domains I-VI in pink
and domain VII in purple. Domain VII wraps around the left monomer and fills in a cavity that leads directly
to the active site of this neighboring monomer. b) PFOR is shown as a gray semitransparent surface, with
TPP and pyruvate shown as sticks, and Mg" shown as a green sphere. When domain VII'(from the opposite
monomer) is omitted from the model, a direct solvent channel to the active site of PFOR opens up, which
is a likely location for CoA binding.
57
a.
V-4
Vi
N1430.'
R1
OOR
E15
165y
14
b.
e
TPF
Vil'
bl120
PFOR
pyruva
T31
1559
V553
Figure 2.10. Channel to the active sites of OOR and Da PFOR are plugged by different structural features.
a) The channel to the OOR active site, running between domains III, I, and VI is occupied by an unstructured
loop inserted into domain III between Alal 44y and Gly 1 65y. Glu 1547 makes a salt bridge to Arg3 1 a, which
in turn interacts with the substrate binding pocket. Notable close interactions are indicated by dashed lines.
b) Access to the active site in Da PFOR is shown in the same orientation as (a), with the same coloring
scheme for domains I - VI. Domain VII', which occupies this channel in the structure of PFOR, is colored
in purple. In this structure, Met1202' interacts with the pyruvate-binding site, providing a floor to the active
site. These close contacts to Metl202' are indicated by dashed lines, which also indicate other notable
interactions, as in (a).
58
though 50% larger in PFOR than in OOR, maintains a core ferredoxin fold and cluster environment
in both enzymes. Interestingly, OFORs have a wide variety of insertions and deletions in their
ferredoxin domains, with hundreds of enzymes that have minimal ferredoxin domains like that
of OOR. Since these variably sized insertions do not disrupt the 'core' fold, it is likely that these
modifications may have evolved to affect intermolecular protein-protein interactions within the
cell, such as would be required between the OFOR and its cognate redox partner protein. More
work, however, must be done to ascertain the reduction potentials of the OOR clusters to understand
better how these domains function.
In addition to the position of the redox cofactors and environment of the ferredoxin clusters,
the proximal cluster maintains what appear to be key interactions with nearby residues. Asn143P of
domain VI interacts with both TPP and the proximal cluster, and is poised to interact with substrate
as well. This interaction would place any TPP-bound intermediates within the tertiary coordination
sphere of the proximal cluster, and may have a role in facilitating electron transfer during catalysis.
Arg58y of domain III is functionally conserved in PFOR as Lys459, so that both enzymes have a
positively charged residue interacting with both Asn143
and the proximal cluster. This similarity
between OOR and PFOR structures suggests a possible role for domain III, whose function has
been enigmatic, in modulating the reduction potential of the proximal cluster. Finally, the CAGC
motif that provides two cysteine ligands to the proximal cluster in OOR is conserved as a CXGC
motif in all OFORs (Figure 2.11). Both PFOR and OOR structures show identical positioning of
motif residues in the gap between the proximal cluster and the medial cluster, complete with a
conserved water molecule that sits between the motif and the medial cluster (Figure 2.5b,c). This
water is in position to hydrogen-bond to both the glycine backbone nitrogen of the motif and to a
sulfur of the medial cluster. Thus, CXGC residues contact the proximal cluster directly using the
two cysteines and contact the medial cluster indirectly through this conserved water molecule.
Further work will be required to determine if this conserved water plays a role in mediating electron
transfer between these two clusters.
59
MooreLLa OR
Pyrococcus VOR
HRT A
HTA Q
GPAL... ...
GASL... ...
Thermococcus VOR
HTA Q
GASL... ...
Pyrococcus PFOR
Thermotoga PFOR
Giardia PFOR
Trichomonas PFOR
MooreLLa PFOR
Enterobacter PFOR
DesuLfovibrio PFOR
Thauera KGOR
Thermococcus KGOR
MooreLLa KGOR
Thermococcus IOR
Pyrococcus IOR
HAA
HRL
PGS
NGA
SGA
SGA
SGA
PVW
TAL
STA
PTM
PVM
A
P
P
Q
A
G
S
P
P
S
P
P
GCAT ...
GAPI ...
GESN ...
GETA ...
GETP ...
GETP ...
GETP ...
GDYS ...
GGGT ...
GLGQ ...
PHRG ...
PHRG...
....
...
...
...
...
...
...
...
....
...
...
...
Figure 2.11. Alignment of proximal-cluster-binding residues from domain VI of various OFORs. The
conserved CXGC motif spans enzymes of this family (oxalate oxidoreductase (OOR), pyruvate:ferredoxin
oxidoreductase (PFOR), isovalerate oxidoreductase (VOR), a-ketoglutarate oxidoreductase (KGOR),
and indolepyruvate oxidoreductase (IOR)) across all branches of life. At left, a tree indicates the relation
between each of the enzymes, calculated by maximum likelihood using a Blossom62 model of amino acid
substitution (51), from 125 residues in conserved blocks. Species (with Uniprot accession codes) included
are: Moorella thermoacetica(Q2RI42, Q2RMD6, Q2RH06), Thermotogamaritime (Q56317), Pyrococcus
furiosus (Q51805, Q51802, 16V0T7), Thermococcus litoralis (H3ZKIO, H3ZPH4, H3ZL62), Thauera
aromatica (Q8RJQ9), Giardia intestinalis (Q24982), Trichomonas vaginalis (Q27089), Enterobacter
agglomerans (P19543), and Desulfovibrio africanus (P94692).
60
OOR and PFOR operate on very different substrates. Pyruvate is a typical 2 oxoacid, consisting
of one negatively charged acid group, one electrophilic carbonyl, and a hydrophobic methyl
group. Oxalate, on the other hand, is a dicarboxylic acid, which has two negatively charged acid
groups (49), which are both hydrophilic, and neither of which is particularly electrophilic. From
the perspective of TPP, pyruvate is a much better target for nucleophilic attack than oxalate. The
difficulty of activating oxalate for catalysis is seen in the anaerobic oxalyl-CoA decarboxylase
pathway, which first makes use of formyl CoA transferase to activate oxalate to form oxalyl-CoA.
The thioester in oxalyl CoA is more electrophilic than a carboxylate group, making it a better target
for nucleophilic attack by TPP. In aerobic pathways, Mn3+ presumably allows the generation of a
radical species that results in cleavage of the carbon-carbon bond of oxalate, though the mechanistic
details are still murky (50). M thermoacetica, on the other hand, is capable of activating oxalate for
nucleophilic attack without the aid of CoA, a feat that can now be better understood by comparing
the active sites of OOR and PFOR.
The active site of PFOR is tailored for pyruvate-binding (14). The carboxylate of pyruvate
interacts with various hydrogen-bond donors, including a protein backbone amide (Ile30-Thr3 1),
Thr31, Asn996, and Arg114, which also provides an ionic interaction. The carbonyl group of
pyruvate interacts with the imine from TPP as well as Arg 114 (Figure 2.7b). With Arg 114 providing
two hydrogen bonds, a total of six hydrogen-bond donor interactions and one positive charge are
provided by the active site to bind pyruvate. The methyl group is not neglected-PFOR provides
Leul21 and Ile 123 as hydrophobic interactions with pyruvate.
OOR's active site has a number of substitutions relative to PFOR that make it amenable to
binding oxalate. In domain I, Thr31 has been substituted with an arginine, and the structure shows
that Arg31a is positioned for interacting with the substrate-binding pocket. Leul2l and Ie123
in PFOR are substituted by Gly115a and Phe117a in OOR, allowing access to the active site
for an interaction with Gln21 Ia'. Together with TPP and Asn143 P, the OOR active site provides
seven hydrogen-bond donors all pointing toward the presumed substrate-binding site, including
two cationic interactions, to stabilize the binding of oxalate. Thus, the substrate-binding pocket
61
in OOR is more positively charged and hydrophilic relative to PFOR. Beyond substrate binding,
these hydrogen-bond donors may also stabilize resonance structures of oxalate that are more
electrophilic in nature, potentially making it a more favorable target for nucleophilic attack by TPP.
The question of access to the active site is important for PFORs and other OFORs in general
because of the requirement for CoA to complete the catalytic cycle. Although multiple structures
of Da PFOR have been obtained, a structure in complex with CoA remains to be determined. It is
possible that the C-terminal domain VII that is peculiar to the Da enzyme, and which occupies a
central cavity that leads directly to the active site, is blocking the CoA binding site in the crystal.
However, without further evidence, the question of CoA binding to PFOR remains unanswered.
OOR, like other OFORs, does not have a domain VII, so it was hypothesized that the OOR
structure might reveal how substrates of OFORs access the active site. It was a surprise, therefore,
when an extended loop from domain III of OOR (residues 144 165 of OOR y) was observed to fill
the cavity left empty by the lack of a domain VII, completely blocking access to the active site.
This loop links two helices in the C-terminal end of domain III and is 13 residues longer than the
corresponding linker in Da PFOR. Apart from Glul 54y, which forms a salt bridge to Arg3 1 a in the
active site, this loop makes no substantial interactions with the surrounding protein. This extended
loop is conserved only in enzymes with greater than 65% identity to M thermaceticaOOR, so it
does not appear that this loop will be a hallmark of OORs. It does, however, show how predictions
of substrate channels or solvent accessibility is challenging in this family of enzymes, with the
potential for simple insertions at loop positions to substantially alter the active site environment.
Conclusion
Structures of OOR and PFOR show similarities where we expected differences and differences
where we expected similarities. Despite the fact that the catalytic unit of Da PFOR consists of
one peptide chain with seven domains, the trimeric structure of the OOR catalytic unit with five
domains is quite similar in overall architecture. We see how a trimmed ferredoxin domain can
house an almost identical arrangement of iron-sulfur clusters, and how a loop can replace domain
VII to seal a channel to the active site. These structural data also show how substitutions in an
62
OFOR active site can allow for activation of a relatively nonreactive substrate, in this case oxalate.
Overall, the structural similarities and differences between OOR and PFOR provide a framework
for understanding how nature harvests the low-potential electrons that are essential for fundamental
processes such as nitrogen and carbon fixation, and they serve to inform about the larger family of
OFOR enzymes that are pervasive throughout microbial life.
Acknowledgments
This work was supported in part by National Institutes of Health Grants GM069857 (C. L. D.)
and GM39451 (S. W. R.), and by the National Science Foundation (NSF) Graduate Research
Fellowship under Grant No. 1122374 (M. I. G.). This research was also made possible by the
support of the Martin Family Society of Fellows for Sustainability (M. I. G.). For more information
about the fellowship please visit http://dusp.mit.edu/epp/project/martin-family-society-fellowssustainability. C. L. D. is a Howard Hughes Medical Institute Investigator. This work is also based
upon research conducted at the Advanced Photon Source on the Northeastern Collaborative Access
Team beamlines, which are supported by a grant from the National Institute of General Medical
Sciences (P41 GM 103403) from the National Institutes of Health. This research used resources of
the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility
operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DEAC02-06CH11357.
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48. Astashkin AV, Seravalli J, Mansoorabadi SO, Reed GH, Ragsdale SW (2006) Pulsed Electron
Paramagnetic Resonance Experiments Identify the Paramagnetic Intermediates in the Pyruvate
Ferredoxin Oxidoreductase Catalytic Cycle. JAm Chem Soc 128(12):3888-3889.
49. Haynes WM ed. (2015) CRC Handbook of Chemistry and Physics (CRC Press/Taylor and
Francis, Boca Raton, FL). 95th Ed.
50. Whittaker MM, Pan H-Y, Yukl ET, Whittaker JW (2007) Burst Kinetics and Redox
Transformations of the Active Site Manganese Ion in Oxalate Oxidase. J Biol Chem
282(10):7011-7023.
66
Chapter 3
Intermediate-bound structures of OOR reveal enzyme functionality driven by active-site
rearrangement
Summary
Two structures of OOR in the presence of substrate have been determined: one from a crystal
soaked in an oxalate solution, the other from a crystal grown in the presence of oxalate. Electron
density for an oxalate derivative bound to TPP as well as a CO 2 derivative bound to TPP is observed
in different active sites, which is consistent with proposed catalytic intermediates. In addition
to these TPP adducts, significant rearrangements of an active-site loop suggest that the protein
is playing an important role in driving oxalate oxidation by altering the active-site environment
mid-turnover. Specifically, Arg3 1 a that is proposed to facilitate oxalate binding is observed in
an alternate conformation that is swung away from the active site. At the same time, Asp1 16a,
which previously had been facing away from the active site, flips around to come directly into
contact with substrate. We believe that these active-site movements, as well as substrate access
to the active site, are facilitated by movement of domain III and the disordering of a "plug loop."
Together, these observations suggest that OOR operates via a bait-and-switch mechanism, luring
substrate into the active site, and then reorganizing to drive catalysis.
67
Contributors
Aileen C. Johnson, an MIT undergraduate who worked with me, grew the crystals that were
soaked with oxalate. She performed the oxalate soak, as well as looping and cryo-cooling the
crystals for data collection. Edward J. Brignole (MIT) determined initial crystallization conditions.
Elizabeth Pierce (UMich) purified OOR from M thermoacetica, and performed enzyme assays
and ensured the quality of the protein. Mehmet Can (UMich) performed enzyme assays and
ensured protein quality. Stephen W. Ragsdale (UMich) and Catherine L. Drennan provided
invaluable guidance and discussion in planning these experiments and interpreting the results.
68
Introduction
Oxalate is a difficult molecule to metabolize, so much so that humans and most animals lack
the capacity to break it down. This inability to metabolize oxalate is associated with chronic renal
disease in humans, some of the manifestations of which are painful kidney stones, crystalline
arthritis, and total kidney failure (1-3). Plants, fungi, and some gram-positive bacteria have
evolved oxygen-dependent pathways for metabolizing oxalate with the help of manganesedependent enzymes in the cupin family of proteins (4, 5). These aerobic enzymes, oxalate oxidase
and oxalate decarboxylase, take advantage of the propensity of oxalate to ligate metal ions to
form a Mn-oxalate complex, following which dioxygen is employed to generate an oxalate-based
radical species that causes rapid decarboxylation. The reduced products of these enzymes are
hydrogen peroxide in the case of the oxidase and formate in the case of the decarboxylase.
Before 2010, only one enzyme pathway was known to catalyze the degradation of oxalic acid
in the absence of dioxygen: oxalyl-CoA decarboxylase (6). This enzyme is best known for its role
in the metabolism of Oxalobacterformigenes, a bacterium that colonizes the human gut and has
been the subject of research into the role of the human gut microbiome in preventing, and possibly
treating, human renal disease (7-11). As its name implies, however, oxalyl-CoA decarboxylase
first requires the activation of oxalate by coenzyme A (CoA), a feat that is accomplished by a
formyl-CoA transferase. This activated oxalate, featuring a thioester in place of a carboxylate, is
susceptible to nucleophilic attack by thiamin pyrophosphate (TPP; see Figure 1.3), which is the
catalytic cofactor in this anaerobic enzyme. The end product of this reaction is formate, which can
either enter cellular metabolism or be pumped out of the cell by a formate:oxalate antiporter to
generate ATP (8).
Recently, an oxalate oxidoreductase (OOR) was reported that performs an unprecedented
metabolic transformation of oxalate by splitting it into two molecules of carbon dioxide and
recovering two reducing equivalents for use in cellular processes (Figure 3. 1a) (12). This OOR,
discovered in Moorella thermoacetica (13, 14), feeds both carbon dioxide and low-potential
electrons into the Wood-Ljungdahl pathway for acetogenesis, allowing M thermoaceticato grow
69
-0
a.
0
+ 2 FdOX
0
C.
OOR
00
0
0+ -SCoA + 2 Fdox
/"PFOR
-
- C2 +
+ 2 Fdred
)
b.
0
v- 2 C0 2 + 2 Fdred
SCoA
d.
PFOR
is
e.
Figure 3.1. OOR and PFOR use TPP to catalyze the oxidation oxalate and pyruvate respectively, capturing
the electrons in [4Fe-4S] clusters, a) The reaction scheme for OOR. b) The reaction scheme for PFOR. c)
The catalytic dimer of OOR is shown as a ribbon diagram. The right monomer is colored purple, while the
left monomer is colored by domain: I - green; II - blue; III - yellow; V - wheat; VI - red. Buried [4Fe-4S]
clusters are shown as ball-and-stick models, and TPP is shown as white sticks. d) The catalytic dimer of
PFOR (PDB 2C42) is shown as in c), but with the right monomer shown in gray. PFOR has two additional
domains, IV (orange) and VII (pink). e) The cofactor arrangement for QOR and PFOR are shown overlaid
onto each other. OOR is shown as in c), while PFOR is colored gray. Dashed lines indicate electron-transfer
distances.
70
exclusively on oxalate-a boon to this anaerobic bacterium commonly found in soil, which is
often rich in oxalate (15). Like oxalyl-CoA decarboxylase, OOR uses TPP as its catalytic cofactor.
OOR is unique, however, in that it does not require CoA to activate oxalate, and it uses a chain of
[4Fe-4S] clusters to store and transfer the electrons captured from the C-C bond.
OOR belongs to a larger superfamily of microbial enzymes, known as 2-oxoacid:ferredoxin
oxidoreductases (OFORs), which use TPP and [4Fe-4S] clusters to oxidize pyruvate and other
cellular building blocks (16-19). The best characterized family member is pyruvate:ferredoxin
oxidoreductase (PFOR), for which X-ray structures (Figure 3.1d) and some biochemistry are
available for the Desulfovibrio africanus enzyme (Da PFOR) (20-23), and more biochemistry
and spectroscopy are available for the Moorella thermoaceticaenzyme (24-26). PFOR catalyzes
the reversible cleavage of pyruvate to form C0 2, acetyl-CoA, and two electrons that are capable
of reducing low-potential ferredoxins (Figure 3.1b). In this reaction, TPP acts as a nucleophile
and forms a covalent adduct with pyruvate to initiate the subsequent decarboxylation. Although
we assume the same will be true for OOR, oxalate is a very different substrate than pyruvate
and the other 2-oxoacid substrates of the OFOR superfamily, all of which are characterized by a
ketone immediately adjacent to a carboxylic acid. Thus, all substrates, except oxalate, are typically
monoprotic acids carrying one negative charge at neutral pH. Oxalate, on the other hand, is a
diprotic acid and carries two negative charges at neutral pH (27), and is therefore less amenable to
nucleophilic attack by a TPP cofactor.
To probe how OOR was adapted to act on oxalate, we previously solved the structure of OOR
from M thermoaceticain its resting state (Chapter 2) (28). Structural analysis revealed that OOR is
a dimer of trimers, (aylp) 2 , with each ayl forming a catalytic unit that is composed of five domains
(Figure 3.1c). The active site, which houses the TPP and one of the three [4Fe-4S] clusters, is
formed by domains I and VI and appears to be more polar than that found in PFOR, consistent
with OOR's unique substrate. Domain V binds the other two [4Fe-4S] clusters and is the conduit
for electrons to reach the protein surface where they can reduce a ferredoxin (Figure 3.1 e). In this
chapter, we further interrogate substrate binding to OOR through soaking and co-crystallization
71
experiments with oxalate. In addition to identifying residues responsible for activating oxalate,
these structures allow us to consider the molecular mechanism responsible for release of each
molecule of the oxalate-derived CO 2 from TPP. Also, the previously determined OOR structure
(Chapter 2) showed a closed active site, raising the question of how oxalate gains access to the
active site and how CO 2 is released. Here, structures reveal conformational changes that appear to
gate substrate and product access.
Materials and methods
ProteinPurification
OOR was purified from M thermoacetica by the methods described (12), concentrated to
45 mg/ml, as determined by the rose bengal method (29) with a lysozyme standard and was
stored in a storage buffer of 50 mM Tris pH 8.0 and 2 mM dithiothreitol under an anoxic nitrogen
atmosphere. Aliquots of 200 to 500 tL were flash-frozen in liquid nitrogen for long-term storage
in liquid nitrogen.
Crystallization
OOR was crystallized by the hanging drop vapor diffusion method at room temperature in a
Coy anaerobic chamber under an Ar/H 2 gas mixture, as described previously (28) and in Chapter 2.
Briefly, 30 mg/ml OOR in the storage buffer (50 mM Tris pH 8.0 and 2 mM dithiothreitol) was
mixed with the well solution (8-11% PEG 3,000, 3-4% Tacsimate, pH 7.0) in a 1:1 ratio to make a
2 d hanging drop with a final protein concentration of 15 mg/ml. Tetragonal crystals in the space
group P43 grew in 2-4 days. One of these crystals was soaked for 2 minutes in a solution mimicking
the mother liquor but with the addition of 100 mM oxalic acid (Sigma-Aldrich) solution, pH 8.0
(the pH was adjusted with 10 M NaOH), to a final concentration of 10 mM oxalic acid, 15% PEG
3,000, 2% Tacsimate pH 7.0, 25 mM Tris pH 8.0, 1 mM dithiothreitol, and 20% PEG 400, which
served as a cryoprotectant. After soaking, the crystal was cryo-cooled in liquid nitrogen.
Oxalate co-crystals were grown in an anaerobic chamber in the same condition as described
above, in drops composed of 1 pl well solution and 1 pl OOR (30 mg/ml), but with the addition of
0.2 tL of 100 mM oxalic acid, pH 8.0 to the crystallization drop. Long, rod-like crystals grew in
72
2-4 days, in the space group P212 12 1 with unit cell constants a = 113.6 A, b = 144.1 A, c = 161.7 A.
One of these crystals was cryo-protected using PEG 400, in a solution containing: 20% PEG 400,
15% PEG 3,000, 2% Tacsimate pH 7.0, 25 mM Tris pH 8.0, and 1 mM dithiothreitol. Due to the
fragility of the crystals, this cryosolution was added directly to the crystallization drop in two
successive additions of 1 pl and removal of 1 pl from the opposite side of the drop, followed by a
final addition of 2 pl. The drop was allowed to equilibrate for 2 minutes after each addition. After
-
the final addition, crystals were looped and cryo-cooled in liquid N 2
Data Collection and Processing
Data were collected at the Advanced Photon Source on Northeastern Collaborative Access
Team beamline 24-ID-C on a Pilatus 6MF detector. HKL 2000 (30) was used to process all of the
datasets (Table 3.1). Data for the oxalate-soaked crystal and the oxalate co-crystal extended to
2.50-A and 1.88-A resolution, respectively. The oxalate-soaked crystal was determined by Phenix
Xtriage (31, 32) to have merohedral twinning, with a twin law of h, -k, -1. The twin fraction refined
in Phenix Refine (33) to 35%, and the structure was refined to account for twinning.
Structure Determinationand Refinement
The structure of OOR, described in Chapter 2, was used as a search model for molecular
replacement (MR) in each dataset, which was performed with Phenix (Phaser-MR) (34). MR was
necessary for the determination of both structures because both crystals had different unit cells
than the high resolution native structure, which crystallized in the space group P2 12 12 1 with unit
cell constants a = 84 A, b = 152 A, and c = 172 A. A search model with amino acid sidechains
and cofactors and a resolution cutoff of 3.5 A was used for the MR search in both instances. In
the oxalate-soaked crystal, the asymmetric unit was found to contain two OOR dimers. In the
oxalate co-crystal, the asymmetric unit contained one OOR dimer. Once a MR solution was found,
one round of rigid-body refinement into the full-resolution dataset was performed to verify the
placement of the molecules and improve the electron-density maps. Iterative rounds of modelbuilding in COOT (35) and refinement of atomic coordinates and B-factors in Phenix (33) allowed
for the correct placement of sidechains and loops where they differed from the starting model.
73
Table 3.1. Data Collection and Model Refinement Statistics
Data collection and processing
Space group
Cell dimensions (A)
Wavelength (A)
Resolution (A)
Total unique reflections
Completeness (%)
Redundancy
<I/al>
Rsymt (%)
OOR - oxalate soak
OOR - oxalate co-crystal
P43
a = b = 138.4, c = 217.1
P2 12 12 1
a = 113.6, b = 144.1, c = 161.7
0.97920
50-1.88 (1.91 - 1.88)
200,377
94.4 (75.0)
5.9 (4.1)
0.97900
50-2.50 (2.54-2.50)*
131,808
93.2 (64.5)
4.7 (3.4)
6.8 (1.9)
18.3 (64.1)
(85.0)
CC1 /2 (%)
Model Refinement
R-work (%)
R-free (%)
Protein atoms
18.9
23.1
Oxalate adduct atoms
CO 2 adduct atoms
[4Fe-4S] atoms
Solvent atoms
RMS(bonds) (A)
-
TPP-Mg2+ atoms
30,087
108
6
96
Ramachandran favored (%)
Ramachandran allowed (%)
Ramachandran outliers (%)
386
0.003
0.663
95.7
4.1
0.2
Average B-factor (A 2 )
37.7
RMS(angles) (0)
Protein (A 2 )
TPP-Mg 2* (A 2 )
37.8
32.8
Oxalate adduct (A 2 )
32.5
2
36.8
Water (A 2 )
32.5
-
CO 2 adduct (A )
[4Fe-4S] (A 2 )
*Numbers in parenthesis indicate the highest resolution bin.
ti(II - <I>1)/Y,(I)
74
7.7 (1.9)
12.9 (88.2)
(86.7)
17.7
20.7
17,975
54
6
48
2,225
0.007
1.003
97.0
2.9
0.1
28.8
27.4
19.2
24.4
20.1
40.9
NCS restraints were used in the initial stages of refinement for the oxalate-soaked crystal, though
in later stages these restraints were removed. In each of the structures, potential covalent adducts
to C2 of the TPP ligand were identified by positive difference density in the F0 -Fe maps. Adducts
to TPP were allowed to refine to partial occupancy while holding B-factors constant.
Restraints for resting TPP were based on the crystal structure of a pyruvate decarboxylase (PDB
2VK8). Restraints for an oxalate-TPP adduct, [oxalate-TPP], and a C0 2-TPP adduct, [C0 2 -TPP],
were generated from geometry optimization calculations in Gaussian 03 (36), using a B3LYP
hybrid functional with a 6-31 1++G basis set for all atoms. For the calculations, TPP was trimmed
down to 3,4,5-trimethyl-3k4-thiazole. End-state frequency calculations were performed to ensure a
minimum energy potential had been reached. An unrestricted functional was used for C0 2 -thiazole
calculations, so that all three oxidation states could be compared. The differences in calculated bond
lengths and angles between reduced, 1-electron oxidized, and 2-electron oxidized C0 2 -thiazole
were small enough that they would be indistinguishable at the resolution of the crystal structures.
The calculated bond distances and angles for the oxalate- and C0 2-thiazole adducts were used to
update previously employed TPP restraints. CO 2 was restrained to be planar with the thiazole ring,
whereas the dihedral angle between oxalate and the thiazole ring was allowed a greater degree of
freedom to account for specific interactions with the protein. Restraints for the iron-sulfur clusters
were based on Moorella thermoacetica carbon monoxide dehydrogenase/acetyl-CoA synthase
(PDB 3101) (37), though they were loosened for the higher resolution structures.
In the structures from both the oxalate-soaked crystal and the oxalate co-crystal, residues
113-117 of chain a, which form a loop in the substrate-binding pocket, were disordered to some
extent, with the electron density suggesting two distinct conformations. In the soaked crystal, the
data were sufficient to model one orientation for this loop. In the co-crystal, however, the higher
resolution and clearer difference density made it possible to model this loop in two conformations.
Protein regions that were modeled in two conformations are shown in Table 3.2.
Simulated-annealing composite-omit maps (made with Phenix Autobuild (33)) were used to
validate the final models. For each dataset, residues not having sufficient electron density were
75
Table 3.2. Model completeness* of OOR crystal structures.
OOR - oxalate soak
Chain
A
B
C
D
E
F
G
H
J
K
L
Chain
A
B
C
D
E
Omitted residues
1
1-3, 146-164, 211-218
313-314
1
1-5, 40-44, 211-223
311-314
1
1-4, 145-164
313-314
1
1-5, 145-166, 210-219
311-314
J
K
L
1
1-2, 214-217
1
1, 146-164, 214-218
314
N/A
N/A
N/A
N/A
N/A
N/A
Residues modeled as alanine
3
4,145,191,219,226
3
233,314,383
11, 13, 15, 25, 26, 29, 33, 36, 50, 53, 69-72, 79,
83, 84, 87, 89, 101, 103, 109, 111, 112, 114-124,
126, 128, 129, 131, 132, 134-138, 141, 143, 145,
148, 158, 174, 176, 178, 180, 184-186, 189-192,
196,205,207,208,226,314
383
4,26,145, 176,314
1
G
H
OOR - oxalate co-crystal
11
4
1
''
3, 383
5,6, 191
1,4,267
3, 383
26, 29, 101, 103, 116, 118, 119, 123, 174, 176,
180, 200, 227, 314
N/A
N/A
N/A
N/A
N/A
N/A
Chain
Residues modeled as alanine
A
31, 108-119,210-220
B
C
D
31, 108-119,210-220
E
F
*Chains A, D, G, J have 395 residues. Chains B, E, H, K have 315 residues. Chains C, F, I, L have 314 residues.
76
Table 3.3. Features of the different monomers in the asymmetric unit of the oxalate-soaked OOR crystal.*
P43 (twinned)
Space Group
2.50 A
Resolution
50 mM Tris, pH 8.0,2 mM DTT
Storage Buffer
protein (in storage buffer) + 4-5 % PEG 3,000, 1-2% Tacsimate
Drop conditions
Drop conditions + 20% PEG 400 + 10 mM oxalate pH 8.0 (1-5 minute soak)
Cryo conditions
Monomer 2 (chains D,E,F)
Monomer 1 (chains A,B,C)
Active Site
Active-site omit
density (1 a)
Intermediate,
occupancy
Switch loop
Domain III loop
Domain III crystal
contacts
Active Site
Active-site omit
density (1 a)
Intermediate
Switch loop
Domain III loop
Domain III crystal
contacts
(Disordered density with nothing modeled)
Oxalate, 89%
Asp-in
Disordered
the in position of domain
stabilizing
Contacts
III.
Monomer 3 (chains G,H,I)
Asp-in
Ordered
Contacts stabilizing the out position of
domain III
Monomer 4 (chains J,K,L)
(Disordered density with nothing modeled)
Asp-in
Disordered
Contacts stabilizing the out position of
domain III
(Disordered density with nothing modeled)
Asp-in
Disordered
Not many close contacts
*See text for definitions of "Switch loop," Domain III loop," and "Domain III crystal contacts."
77
omitted from the model or modeled as alanine. Model completeness along with omitted residues is
shown in Table 3.2. Solvent water molecules were generated automatically and refined by Phenix
using a sigma cutoff of 3.5. Prior to refinement, 5.0% of unique reflections from the each dataset
were set aside as test datasets, from which Rfree values are calculated. Different test datasets were
used for each structure due to the fact that each structure was determined in a different unit cell.
Final refinement statistics are shown in Table 3.1.
Results
Soaking OOR crystals with oxalate generates one active site with oxalate bound to TPP and three
active sites with undefined electron density.
A crystal incubated with oxalate, which diffracted to 2.50-A resolution, was found to have two
OOR dimers in the asymmetric unit (Table 3.1, 3.3). In the active site of monomer 2 (see Table 3.3
for a summary of all four monomers), there was positive difference density adjacent to the C2
carbon of the TPP thiazole ring. A model for the [oxalate-TPP] covalent adduct was refined in this
active site, and was found to account well for the electron density (Figure 3.2a).
It was previously hypothesized that oxalate would bind in the pocket adjacent to the C2 carbon
of TPP (Figure 3.3a) (28). From the structure of the [oxalate-TPP] adduct, we see that this is
indeed the case (Figure 3.3b). In addition to forming a covalent adduct to TPP, [oxalate-TPP]
makes a number of hydrogen-bonding contacts with the surrounding protein, including residues
Arg3la (the amide nitrogen as well as the guanidinium amines), Argl09a, and Asn1430. There
is also an interaction with the imine nitrogen of the aminopyrimidine moiety of TPP (as noted
in Chapter 2, the imine tautomer is stabilized by hydrogen-bonding to Ni' by Glu59a; also see
Figure 1.3). Also making a close contact with the oxalate adduct is Asp1 16a, which is part of a loop
from residues 107-121 of chain a that helps form the substrate-binding pocket. In the previously
reported structure (Figure 3.3a), Aspil6a points away from the active site and Phell7a points
toward where substrate would bind (an "Asp-out" conformation). However, in the active site with
oxalate covalently attached to TPP, we see this situation reversed, with Asp 11 6a rotated about 1500
to point toward the carboxylate group of oxalate, and Phe 11 7a rotated about 90' and the phenyl
78
b
Figure 3.2. Stereoscopic views of substrate-bound intermediates in OOR and corresponding simulatedannealing composite-omit electron density maps contoured at 1 a. a) The [oxalate-TPP] adduct (white) and
the sidechains of residues Arg3 Ia, ArgI09a, and Aspi 16a (green) are shown as sticks. The electron density
is shown as a gray mesh. b) The [C0 2 -TPP] adduct from monomer 1 of the oxalate co-crystal is shown with
the same representation as in a), but due to a conformational change of the Switch loop, Phe 1 17a is now
shown instead of Asp I16a.
79
a
N'H
R109a
H-N'+
H
D112a
H
H
~
'N+H
TP
N+
ON -H
R109a
116a
s R
H
N -H
H
H N
HH NN-H_H
F117a
Q211a'
R31a
NH
R109a
N
H-N
109a
0
N1430
N ,H
w
H
H
-
R 3 la
Q211Ia' Q211alN1430
N
H
[oxalate-TPP]
N+
;
D116a
V-NH
,
H-N+
H
H *Nry
0 N1430
H
-
R31a
'N -H ---N mX',+
0
,N -H
R31a
H
VD116a
/
N1430
N-
D112
NH
H
R109a
R1O9a
R2
S
4
- sy
F 1 7a
NH
H
---- 0N - - ---------
C
NH
-
(C0-,.?
N
'
2
R2
S
H' 0,H-'
H
0
Q211a'
N143P
N
H
F117a
[CO2 -TPP]
R109a
H
H-N
-
D116a ,,
F117
R31a
80
0
N1430
H H
H
NH
'c
0
-
-
H~ H
D1l2a
Q211N
H
I
0-.
112a
d
Q211a
Hl
R31a
N
s
R2
R109a
N1430
0
H
D116a
0
N1430
Figure 3.3. Views of the OOR active site at different stages of catalysis, with corresponding Lewis structure
diagrams at right. a) The active site of as-isolated OOR, from the structure previously reported (Chapter 2),
is vacant and ready for substrate binding, with the Switch loop in the Asp 116a-out conformation. b) The
[oxalate-TPP] adduct from monomer 2 of the oxalate-soaked crystal is shown with the Switch loop in the
Asp-in position. c) [CO2 -TPP] is shown from monomer 1 of the oxalate co-crystal, with the Asp-out loop
conformation. Gln2 11 a' is oriented in toward the active site, forming a hydrogen bond through water to
the adduct. d) The active site of monomer 2 of the oxalate co-crystal is shown with [C0 2-TPP] interacting
with Asp 1 6a from the Switch loop in the Asp-in conformation. Gln2 11 a' is pulled away from the active
site and is no longer supporting a water molecule in the same position as in c). In the Lewis diagrams,
protonation states are hypothesized, as is the oxidation state of [C0 2-TPP], which is depicted in the most
reduced state as a methylene diol adduct. Oxygens of the adducts are numbered in the Lewis diagrams in
b-d). In the structure figures, the protein is shown in ribbon diagram with domain I colored green, domain
VI colored red, and TPP adducts colored white (See Chapter 2 for domain definitions). TPP, its adducts, and
sidechains of residues Arg3 1 a, Arg 1 09a, Asp 12a, Asp 116a, Phe 117a, Gln2 11 a', and Asn 143 P are shown
as sticks. Select active-site water molecules are shown as non-bonded red spheres and dashed lines indicate
close distances to TPP bound adducts. In the Lewis diagrams, potential hydrogen-bonding interactions with
bound substrate are indicated by black dashed lines, and interactions that are hypothesized to be chargerepulsion interactions are shown as red contoured lines.
81
group swung away from the active site ("Asp-in" conformation). To enable this rearrangement, a
PPG motif (residues 113-115 of chain. a) that precedes Asp 16a, and forms the hairpin turn in this
loop, folds back away from the active site, allowing rotation of the peptide bonds of Asp I16a and
Phel 17a. Together, these residues form what we will call the "Switch loop" (Figure 3.4).
The other three active sites of the oxalate-soaked crystal, in monomers 1, 3, and 4, also
had positive difference density adjacent to C2 of TPP (Table 3.3), but the electron density was
too disordered to refine a specific adduct with confidence. There was sufficient density in each
of the active sites, however, to model and assign the Switch loop to the predominantly Asp-in
conformation.
Co-crystallizationof OOR with oxalate generates two active sites with CO 2 bound to TPP
The structure of OOR incubated with oxalate during the crystallization process was solved
to 1.88-A resolution, and found to contain one dimer in the asymmetric unit (Table 3.1, 3.4).
In both active sites, positive difference density was observed adjacent to the C2 carbon of the
TPP thiazole ring. This electron density was best modeled as product bound to TPP [C0 2-TPP]
(Figure 3.2b), with CO 2 refined at partial occupancy (60-70%) (Table 3.4). Difference density also
indicated two distinct conformations of the Switch loop in both active sites. Both conformations
of this loop (residues 108-119 of chains A and D) were modeled and refined with split occupancy
(Figure 3.5). In the active site of monomer 1, the Asp-out conformation is predominant, whereas in
the other active site, monomer 2, the Asp-in conformation is in the majority. Therefore, two active
sites provide two different sets of environmental interactions with the bound CO 2 intermediate. In
monomer 1 (Figure 3.3c), a number of potential hydrogen-bond donors are making interactions
with the bound C0 2, including Argl09a, Asn143P, and Gln2lia', through an ordered water
molecule. In monomer 2 (Figure 3.3d), the ordered water molecule has moved up in the active site
to interact with Aspi 12a, and due to the hydrogen-bonding bond angle (Asp-H 20-[CO 2-TPP] =
1430), is likely not able to donate a hydrogen bond to the bound intermediate. Asp1 16a from the
Switch loop is also nearby the bound intermediate, but is not positioned at an angle that suggests
82
M
a
Asp-out
P113a
P1 14a
G115a
D116a
TPP
F117a
b
t
Asp-in
P113a
P114a
G115a
TPP
F117a
D116a
Figure 3.4. Residues 113-117 of chain a in OOR form a Switch loop that has two alternate conformations. a)
The Asp-out conformation is shown with the Switch residues as stick models. In this conformation, Phel 17a
is oriented toward the substrate-binding pocket. Arrows from Prol13a, Aspi16a, and Phel17a indicate
the direction of movement of these residues to go from Asp-out to Asp-in. b) The Asp-in conformation is
shown as in a). In this conformation, Asp 1 6a is oriented toward the substrate-binding pocket.
83
I
Table 3.4. Features of the different monomers in the asymmetric unit of the OOR oxalate co-crystal*
Space Group
Resolution
Storage Buffer
Drop conditions
Cryo conditions
Active Site
Active-site omit
density (1 a)
Intermediate,
occupancy
Switch loop
Domain III loop
Domain III crystal
contacts
P2 12 12 1
1.88 A
50 mM Tris, pH 8.0, 2 mM DTT
%2 protein (in storage buffer) + 4-5 % PEG 3,000, 1-2% Tacsimate + 10 mM oxalate
Drop conditions + 20% PEG 400 (1-5 minute soak)
Monomer 1 (chains A,B,C)
Monomer 2 (chains D,E,F)
CO 2 (best density), 61%
C0 2, 65%
Asp-out
Ordered
No contacts
Disordered
No contacts
Asp-in
*See text for definitions of "Switch loop," Domain III loop," and "Domain III crystal contacts."
84
a
Monon
'113a
P114a
G115a
6c;
902-TPP]
F117a
b
P113a
P114a
G115a
D116a
QCO 2-TPP]
F117a
Figure 3.5. Electron density in the oxalate co-crystal shows that both Switch loop conformations are
present at different occupancies. a) The Switch loop of monomer 1 is shown in both conformations in stick
representation. Simulated-annealing composite-omit maps contoured to 1 a are shown as a gray mesh. The
Asp-out conformation is dominant in this monomer (62%), though there is clearly density for the alternate
conformation as well. b) The Switch loop of monomer 2 with simulated-annealing composite-omit maps
shown as in a). Again, density is visible for both loop conformations, though the Asp-in conformation is
dominant (58%).
85
that it makes a hydrogen-bonding interaction. Instead, Asp 116a may engage in a charge-repulsion
with the bound intermediate.
In addition to direct interactions with the active site, the Switch loop interacts with the
C-terminus of domain I. In the Asp-in conformation, Phe17a interacts with Pro2l4a', which is
part of an extended portion at the C-terminus of domain I of the opposite monomer (Figure 3.6).
The rotation of Phe1 17a out of the active site causes Pro2l4a' to shift by about 2.5
A, which results
in an alternate conformation for residues 210-220 of chain a. These two distinct conformations
were apparent in the difference density, and so are modeled with split occupancy. In monomer 1,
which is primarily in the Asp-out conformation, Gln211 a' is seen interacting through an active-site
water molecule with the TPP-bound CO 2 intermediate as described above (Figure 3.7a). However,
in monomer 2, which is in the Asp-in conformation, Gln211a' has been pulled away, and the water
has moved up to interact with Asp 112a as well as the proposed CO 2 intermediate (Figure 3.7b).
Additionally, this moving water molecule becomes connected to an extended hydrogen-bonding
network up through the active site in the Asp-in conformation (Figure 3.8).
Q211a'
Q211a'
P214a
P214a
D116a
[C0 2-TPP]
D116a
[C0 2-TPP]
Figure 3.6. Stereoscopic view of loop movement in the active site of monomer 2 of the oxalate co-crystal.
Alternate conformations of loops containing residues 113-118 of chain a and residues 210-220 of chain a'
are shown, with noted active-site residues shown in sticks. Residues belonging to the Asp-in conformation
are shown in green with colored heteroatoms, whereas residues in the Asp-out conformation are colored
white. In the Asp-in conformation, Phe1 17a interacts with Pro2l4a', pushing it up and away from the active
site. At the same time, Asp 1 16a blocks Gln21 Ia', and directs it away from the active site.
86
112
2r12
sr us3.2
[COT
2
Q211a'
TPP
n
t
Q211a'
112
12
8
,.3.
.3.4
,,R109a
2.3
Q211a'
8
,X
.3.0
33.
02
TPP]
81090
.3.4
0 2TPP]
'.3'
211a'
Figure 3.7. Movement of Gln21l' and active-site water arrangement is correlated with the conformation of
the Switch loop in the oxalate co-crystals. a). The active site of monomer I is shown in stereo. The Switch
loop is in the Asp-out conformation, and Gln21 Ia' is pointed toward the substrate-binding pocket. Activesite residue sidechains and the [C02-TPP] adduct are depicted as sticks and waters as non-bonded spheres.
Simulated-annealing composite-omit density is shown in pink mesh around the water molecules. Contacts
within hydrogen bonding distance to the water molecules are indicated with dashed lines, with distances
given. Residues making contacts to the depicted waters are labeled. b). The active site of monomer 2 is
shown as in a). The Switch loop is in the Asp-in conformation and Gln2 11 a' is pulled away from the active
site.
87
W
E9)Oa
T750
E9Of
T75P
a
D112a
D112a
174P
174P
50P
T0SORP9
R10a
[CO 2-TPP]
,
Q211a'
b
Q211a'
V
T750
EO'
174P
D112a
R10
T50
[CO2-TPP]
D117a
[C0 2-TPP]
T75P
E907'
74P
D112a
R109a
T5Op
,
[C027TPP
D117a
Figure 3.8. Stereoscopic views of the extended hydrogen-bonding networks in the oxalate co crystal. a)
The active site of monomer 1 is shown with [C0 2-TPP] and residue side chains shown as sticks, waters
shown as non-bonded spheres, and composite-omit maps around the waters as a pink mesh at 1.5 a cutoff.
Relevant interactions indicated with dashes. b) The active site of monomer 2 is shown in the same manner
as a). The water that interacts with Gln21 Ia' in a) moves up to interact with Asp1 12a in b) to join a water
network up through the active site.
88
M
[oxalate-TPP]
3.5,
R31 a
)
83.4
El154y
Figure 3.9. Glul 54y from the domain III plug loop forms a salt bridge to Arg3 lIa in monomer 2 of the
oxalate soaked-crystal. Arg3 1Ia in turn forms two hydrogen-bond-donor interactions with [oxalate-TPP],
one with the backbone amide nitrogen, the other with the guanidinium moiety. Domain I is colored green,
domain VI is colored red, and the plug loop is colored bright orange. Hydrogen-bonding interactions are
indicated with dashed lines, along with distances.
89
Arg3l and the domain IH plug loop appear to play a role in product release.
Arg31a is observed in two distinct conformations relative to the substrate-binding pocket
(Table 3.3; Table 3.4; Figure 3.3). Prior to and after oxalate is bound to TPP, Arg3 Ica is positioned
to make hydrogen-bonding interactions with oxalate. (Figure 3.3a,b). In active sites modeled with
[C0 2 -TPP] however, Arg3 Ia is in two conformations: one where it is positioned at the bottom of
the substrate-binding pocket (as above, and in Figure 3.3c) and one where it has swung away from
the active site such that is well away from the C0 2 -TPP adduct (Figure 3.3d). This swung-out
conformation of Arg3 1 a is also seen in other monomers of the soaked crystal, in which TPP was
modeled without an adduct. As was noted previously (28), Glul54y on a loop from domain III,
forms a salt bridge to Arg3 1 a in the active site. When nothing is bound to TPP and immediately
after oxalate binding, this interaction helps to poise the guanidinium moiety ofArg3 1 a in the active
site to interact with substrate (Figure 3.9). This loop, containing residues 146y-163y, occupies a
channel between domains that leads directly to the active site, thus preventing solvent access to the
active site in the state previously reported (Figure 3.1 Oa). Because of this role in sealing the active
site, we will refer to this loop as the "plug loop."
When Arg3 1 a is observed swung away from the active site, Glul 54y and the plug loop are
no longer visible in the electron density, suggesting that they have enhanced flexibility. Further,
these movements are accompanied by a large movement of domain III, which rotates outward
such that the terminal connectors of the plug loop are pulled away from the active site by up to
5 A (Figure 3.11). Together, the movements of the domain III and Arg3 1 a appear to both open and
close the active site (Figure 3.10).
Discussion
Oxalate is a challenging molecule to metabolize. In the previous chapter, we hypothesized that
the addition of polar residues to the OOR active site, as compared to Da PFOR, would play a role in
preparing it for nucleophilic attack by the deprotonated C2 carbon of TPP. The structure of oxalate
bound to the TPP active site supports this hypothesis (Figure 3.12). In both OOR and PFOR, the
carboxylate group of the TPP adduct makes hydrogen-bonding interactions with residue 31 (Arg3 1 a
90
M
Figure 3.10. Solvent access to the active site is gated by the domain III "plug loop". a) The plug loop is
ordered in monomer 1 of the oxalate co-crystal. [C0 2-TPP] and the proximal [4Fe-4S] cluster are shown
buried underneath the protein surface. Domain III is colored yellow. b) Domain III is rotated away in
monomer 2 of the oxalate co-crystal, causing the plug loop to be disordered. Together with Arg3 la being
rotated away from the active site, a channel to the [C0 2 -TPP] is visible.
91
'"0
______________
G0u54
Figure 3.11. Domain III can swing away from the active site, opening a channel for solvent access.
Domains I (green), II (blue), III (yellow), and VI (red) from monomer 1 of the oxalate co-crystal are as
shown as ribbons. The plug loop of domain III of monomer 1 (residues 146y-163y) is colored bright orange
for emphasis. Monomer 1 is overlaid onto monomer 2 of the oxalate co-crystal, shown as gray ribbons.
Whereas the other domains align perfectly, domain III shifts by up to 5
A between the two monomers,
and
this rotation away from the active site is associated with the disordering of the plug loop that occupies a
channel to the active site and makes a salt-bridge via Glul 54y to Arg3 Ia. Since there is no electron density
to confidently place the plug loop in the rotated-out conformation, it has been omitted from the model.
92
R10a
[oxalate-TPP]
16a
-
MOOR
'
N143P
R31a
4IN
L121
1123
R1 14
[pyruvate-TPP]
ft
N996
T31
1123
DaPFOR
M1202
Figure 3.12. Oxalate forms an adduct to TPP in OOR analogous to the pyruvate-TPP adduct in PFOR. a)
The active site of monomer 2 of the oxalate-soaked crystal is shown as in Figure 3.3, with close (< 3.5 A)
interactions indicated with dashed lines. b) The active site of chain A of PFOR (PDB 2C3P) is shown as in
a), but with carbons colored white.
93
in OOR; Thr31 in PFOR) as well as with the conserved Arg residue (Argl09a in OOR; Argi14 in
PFOR). The oxo-group in PFOR and the analogous oxo group in OOR (01 in Figure 3.3b) interact
with the conserved Arg residue as well as the imine group of the TPP iminopyrimidine. Asn996 in
PFOR makes a hydrogen bonding interaction with the carboxylate of the bound pyruvate (Figure
3.12b), but in OOR Asn143p is farther away (~3.8 A) from the bound oxalate moiety. The biggest
difference is that OOR has a loop (the Switch loop) that changes orientations, bringing Asp 11 6a
into and out of the active site. PFOR does not have an alternate conformation of this loop, and
Leul2l and Ile 123 are providing hydrophobic contacts for the methyl group of pyruvate. In OOR,
02 of oxalate is the equivalent atom of the methyl group of pyruvate, and there are no clear
interactions between 02 of the [oxalate-TPP] adduct with the switch loop in the Asp-in position.
However, when the loop is in the Asp-out conformation, this oxygen atom would be within range
of the active-site water molecule hydrogen-bonded to Gln2l a'. It is possible to see, therefore,
that in addition to drawing oxalate into the active site with myriad H-bond donors, the active-site
functional groups are also capable of stabilizing the intermediate [oxalate-TPP] adduct in a similar
conformation as seen for the [pyruvate-TPP] adduct bound in PFOR.
Once the adduct is formed in OOR, however, all the positive charges and hydrogen bonds
from the surrounding residues stabilize the ground state, resulting in a higher energy barrier for
decarboxylation. To achieve enzyme turnover, therefore, one would predict that this intermediate
state must be destabilized, and OOR appears to have a mechanism to achieve destabilization in
the form of the Switch loop. One of the biggest surprises from these structures is the dramatic
movement of the active-site Switch loop relative to the as-isolated structure presented in Chapter 2.
When substrate is bound and this loop is in the Asp-in conformation, the Asp sidechain comes
within 3.5 A of the TPP-bound intermediate, and when substrate is bound and this loop is in
the Asp-out conformation, Gln2 11a' is positioned to make a favorable through water hydrogen
bond to the TPP-bound intermediate. This conformational change was unexpected because no such
movement was seen in a series of resting-state as well as mid-turnover structures of Da PFOR
(21-23). The Da PFOR loop resembles the Asp-out conformation of the Switch loop (Figure 3.13)
94
and contributes hydrophobic residues to interact with the methyl group of pyruvate. It is unclear at
this time whether this loop in Da PFOR will undergo a conformational change, and if so, whether
this change will be catalytically relevant. For OOR, however, given the drastic change in the
electrostatics of the active site caused by this loop flip, it seems very likely that this conformational
change has a role in OOR catalysis.
In particular, the close proximity of Asp 1 16a to the intermediate adduct species may be driving
decarboxylation and transfer of electrons to the [4Fe-4S] clusters in OOR. This hypothesis is
consistent with a number of observations about decarboxylation reactions that have been made
over the years. Regarding the protein environment for enzymatic decarboxylation, it has been
observed that environments that facilitate decarboxylation are either hydrophobic (stabilizing the
production of hydrophobic C0 2) (38) or negatively-charged (destabilizing the negatively-charged
carboxylate (39, 40)). Computational studies have also shown that adjacent negative charges in
protein environments can accelerate decarboxylation reactions by up to 101 (41). Decarboxylationby-adjacent-negative-charge is a simple and elegant mechanism that relies on the fundamental
principle of charge-charge repulsion, and it is no surprise that enzymes make use of this principle
to accomplish their goal.
The above decarboxylation mechanism raises the question of how it is that we observe oxalate
bound to TPP in one of the active sites in our oxalate-soaked structure -
with the Switch loop
flipped in, one would expect that decarboxylation should have taken place. In this light, it is
important to mention that only one ofthe six active sites formed by soaking and/or co-crystallization
has oxalate bound. The others have undefined density or density consistent with C0 2, indicating
that turnover has taken place. We believe that our ability to observe oxalate in molecule 2 of the
soaked structure is due to a combination of two factors. First, lattice contacts appear to stabilize
a closed "in" position of domain III (Figure 3.14b) that should stabilize oxalate in the active site,
preventing or at least hindering decarboxylation. Second, OOR in the crystals was only allowed
to react with oxalate for a short time (1-5 min) before plunging the crystals into liquid nitrogen.
With chemistry potentially slowed by lattice contacts, this relatively short soak time, followed by
95
G115a
D116
L121
TPP
S122
F117a
12
Figure 3.13. The Asp-out conformation of the OOR Switch loop is analogous to the conformation of the
same loop in Da PFOR. Domain I from both OOR (monomer 1 of the oxalate co-crystal) and chain A of
Da PFOR (PDB 2C3P) is shown in ribbon diagram, with the two enzymes overlaid onto each other. The
[C02 -TPP] adduct for OOR and the [pyruvate-TPP] adduct for PFOR are shown as sticks. The Switch loop
in OOR is colored green, whereas everything else is colored white. In PFOR, Leul21 and Ilel23 form a
hydrophobic interaction for the methyl group of pyruvate. In PFOR, AspI 1 6a and Phe 1 7a are capable of
flipping to bring AspI 16a into contact with the TPP-bound intermediate.
96
rapid cryo-cooling, is likely to be responsible for the observation of bound oxalate. It is important
to note that oxalate is not observed when domain III is stabilized in an open "out" position (Figure
3.14a) or is free to move within the crystal lattice. In all other active sites, domain III is free to
move or stabilized in an "out" position (Figure 3.14a) with the channel open, and oxalate is not
observed. These structural findings are consistent with active site access being regulated by domain
III and the plug loop. Presumably oxalate enters the active site when domain III is swung away and
the plug loop is mobile. Only under these conditions is access to the active site available (Figure
3.1 Ob). With oxalate bound, Arg3 1 a moves in to hydrogen-bond to oxalate (Figure 3.9), ordering
Glul 54y of the plug loop, which forms a salt-bridge to Arg3 1 a. Collectively, these interactions
stabilize a 'closed' position of domain III. The fact that we see CO 2 bound to TPP in some active
sites, on the other hand, is unlikely due to domain III movement. If one CO 2 can leave the active
site, it seems reasonable that the second CO 2 could as well. Instead, the presence of CO 2 is more
likely due to the oxidation state of the enzyme in the crystal. With no electron acceptor, turnover
a
b
Figure 3.14. Lattice contacts can stabilize domain III in either the "out" position or the "in" position. a) In
monomer 1 of the oxalate-soaked crystal, domain III is stabilized by lattice contacts in the "out" position
and the plug loop is disordered. Monomer 1 is colored by domain, whereas other molecules in the unit cell
are colored dark gray. The red star indicates the lattice contact, which is depicted in greater detail in the
inset. b) In monomer 2, lattice contacts prohibit domain III from rotating out from the active site. The red
stars indicate lattice contacts, which are depicted in greater detail, including potential hydrogen-bonding
interactions, in the insets.
97
in the crystal will result in a fully reduced state of the TPP and of the [4Fe-4S] clusters. With no
place for electrons to go, CO 2 should remain attached to the TPP.
With these collective structural observations, we can therefore propose a "bait-and-switch"
mechanism for OOR catalysis (Figure 3.15), with TPP-bound intermediates based upon those
previously proposed (see Figure 1.7) (12,42). In order to bind oxalate and stabilize it for nucleophilic
attack by TPP, the OOR active site has been tailored to provide a number of positive charges
and hydrogen-bond donors (Figure 3.15, state 1). This tailoring includes the specific positioning
of Arg3 1 a by the extended plug loop from domain III, which is not present in other OFORs.
Once the [oxalate-TPP] adduct is formed, however, the active site needs to be rearranged in order
to drive the first of two decarboxylations. The Switch loop flips from the Asp-out to the Asp-in
conformation placing a negative charge in direct contact with the [oxalate-TPP] intermediate,
and pushing Gln2 1a' away from the active site, allowing the water molecule to move up in the
active site. Arg3 1 a moves away from the substrate-binding pocket, removing one of the stabilizing
positive charges, as the plug loop and domain III rotate out. Together, these motions create a
net -2 charge difference between this catalytic state and the substrate-binding state. This charge
difference is likely responsible for driving decarboxylation of [oxalate-TPP] (Figure 3.15, state 2).
After the first decarboxylation step, OOR must navigate its way through a series of [C0 2-TPP]
intermediates that, unfortunately, are indistinguishable at the resolution of the structure. Prior to
oxidation, the adduct to TPP will be a methylene-diol. The imino group of the iminopyrimidine
moiety of TPP will likely have an acidic proton, which is removed from C2 of the TPP thiazole
to activate the cofactor for catalysis (43). The oxygen atom closest to the imino group (01 in
Figure 3.3c,d), will likely pick up this acidic proton (Figure 3.15, state 3). The other oxygen atom
(02 in Figure 3.3c,d), however, is not in close contact with any general acids and is likely to
remain deprotonated. Species 3 must lose an electron to generate a radical [_C0 2 -TPP] intermediate
(Figure 3.15, state 4). Charge-charge repulsion from Asp 11 6a in the Asp-in conformation may
once again help to overcome the barrier for this first oxidation.
98
A likely structure for the radical [-CO2 -TPP] intermediate in shown in Figure 3.15, state 4. This
intermediate is analogous to the proposed state of the hydroxyethyl-TPP [HE-TPP] adduct in PFOR
(See Figure 1.8, state 4) (42). Such neutral species are thought to have a high barrier for electron
transfer. In particular, this radical species in PFOR is long-lived in the absence of CoA (44), and
electron transfer to the [4Fe-4S] clusters happens slowly. Upon CoA binding, however, the second
electron transfer is accelerated 100,000-fold, and acetyl-CoA is eliminated as the reaction product
(25). One explanation for how CoA binding to PFOR accelerates the second electron transfer
step is that the additional negative charge brought to the active site by the deprotonated CoA thiol
drives down the reduction potential of the [HE-TPP] radical, allowing the electron to move into
the low-potential [4Fe-4S] clusters (25). Whether this happens before or after nucleophilic attack
by CoA, however, is still a matter of investigation (42).
In contrast to the slow rate of oxidation of the neutral intermediate in PFOR in the absence of
CoA, the electron transfer from the analogous intermediate state in OOR appears facile, as judged
in vitro by the fact that OOR's [4Fe-4S] clusters are reduced with no TPP-based radical observable
within 30 seconds of initiating OOR catalysis (12). Thus, it is tempting to propose that oxidation of
this neutral species in OOR could be driven by charge-charge repulsion from Asp 11 6a in a manner
that may be analogous to the binding of a negatively charged CoA in PFOR, though without the
formation of a covalent adduct.
Although Asp 116a may faciliatate the first decarboxylation step and the subsequent electron
transfers, once the protonated TPP-carboxylic acid intermediate is reached (Figure 3.15, state 5)
interaction with Asp 11 6a will likely raise the pKa of the bound carboxylic acid, hindering the final
deprotonation and subsequent decarboxylation. Therefore, we propose a second rearrangement
of the Switch loop back to the Asp-out conformation, as seen in monomer 2 of the oxalate cocrystal. This movement not only removes the negatively charged Asp1 16a, but allows the activesite waters to rearrange and form a network once again to Gln21 1Ia'. By donating H-bonds to
the carboxylic acid, this active site arrangment would lower the pKa, allowing the imino group
99
H
N-
- H ,H
N-H.
-
0
-O
R31la
*
HN
S R3
R2
0
HN
H
s
N-H
-
H
R2
S
NH
N
,H
N
H-N IH + H
2)
N+
SHOH
R31a
NH
H.NH
H.,
H -N"+
H
R109a
NH
'
R109a
C
+ N-H
0
H
H
I
l-'
D116a
D116a
H
IN
N
HHNH
CO
NH
N
N
eN
N
H
OI
0022
HNN
H
HO
NHH2NNH
+H NN
O
N+
HO
N+
0
--
N-
S
O
S
/H
b
R109a
H
NH
e
I
H®
NH
N+
Rl09a
N
+
LH
H-N+
H
H
H
,0
S
j
R
H
N-H'
H
R31la
100
H
Q2Hla'
H-N
H
S
R31a
H
H
O
OH
N-H
*
H
HN
N-
H
Q2lla'
H
H N'
H
R2
Figure 3.15. Proposed mechanism for oxalate oxidation by OOR. a) The catalytic cycle showing just
TPP and substrate with intermediates numbered. Surrounding panels show proposed interactions of the
surrounding protein residues with intermediates b) 1, c) 2, d) 3, and e) 5. Oxalate binding to the OOR
active-site and subsequent nucleophilic attack is facilitated by a number of hydrogen-bond donating and
positively-charged residues. After the [oxalate-TPP] adduct is formed, movement of domain III (not shown)
allow Arg3la to swing away from the active-site, which, coupled with the Switch loop flipping to the
Asp-in conformation, causes the net charge of the active site to become more negative. This change, and
especially the interaction between Asp 116a and the adduct, is responsible first for the first decarboxylation
and two subsequent 1-electron oxidations. The final deprotonation prior to the second decarboxylation is
facilitated by Asp 116a flipping back out, allowing Gln2 11 a' to organize a water for hydrogen bonding to
the acid.
101
from the pyrimidine moiety to remove the remaining proton, facilitating the second and final
decarboxylation (Figure 3.15, state 6 to state 1).
Conclusion
Through a series of structures of OOR from M thermoacetica, we have gained insight into
substrate access to the active site, substrate binding and activation by residues in the active site, and
dramatic protein movements that change the charge-profile of the active site and drive catalysis.
These protein motions, which involve a nearly 180' flip of an active-site loop, the rotation of an
entire 200-amino-acid domain, and the disordering of a previously-unknown 20 residue loop that
plugs a channel to the active site, are without precedent in the OFOR superfamily, and as far as
we know, in TPP-utilizing enzymes at large. This mechanism is particularly of interest because
of the difficulty with which oxalate is metabolized in nature. OOR is the only oxalate-degrading
enzyme that does not require either the oxidizing power of dioxygen, or the carbonyl-activating
capabilities of CoA, instead making use of active-site residues to both bind and activate oxalate.
The ability of OOR to function without the aid of CoA appears to be enabled by these substantial
active-site residue rearrangements, and we look forward to the development of a genetic system
for M thermoacetica so that these ideas can be tested by mutagenesis.
With this structural investigation of OOR in hand, we can now consider how OOR fits into
the larger picture of known OFORs and the organisms that make use of them. Additionally, as
Oxalobacterformigenes and purified oxalate decarboxylases are a current focus of potential
therapies for the treatment of oxalate-related disease, Moorella thermoaceticaand OOR open the
door for alternative or even combinatorial therapies. This discussion will be the focus of the next
and final chapter.
Acknowledgments
This work was supported in part by National Institutes of Health Grants GM069857 (C. L. D.)
and GM39451 (S. W. R.), and by the National Science Foundation (NSF) Graduate Research
Fellowship under Grant No. 1122374 (M. I. G.). This research was also made possible by the
102
support of the Martin Family Society of Fellows for Sustainability (M. I. G.). For more information
about the fellowship please visit http://dusp.mit.edu/epp/project/martin-family-society-fellowssustainability. C. L. D. is a Howard Hughes Medical Institute Investigator. This work is also based
upon research conducted at the Advanced Photon Source on the Northeastern Collaborative Access
Team beamlines, which are supported by a grant from the National Institute of General Medical
Sciences (P41 GM 103403) from the National Institutes of Health. This research used resources of
the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility
operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DEAC02-06CH1 1357.
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106
Chapter 4
Oxalate oxidoreductase in context
Summary
With a better understanding of the mechanism by which OOR catalyzes the oxidation of oxalate,
it is possible to take a closer look at some of the important features in the entire family of OFORs,
discuss the function generally performed by these enzymes, and identify other potential OORs.
107
Introduction
In the previous chapters, a number of key catalytic residues as well as substantial protein
motion were identified within oxalate oxidoreductase (OOR) that allow it to convert oxalate to
two molecules of carbon dioxide and capture the two electrons from the C-C bond of oxalate
using three [4Fe-4S] clusters. It is now worth considering the role of 2-oxoacid oxidoreductases
(OFORs) in biology, and specifically the place that OOR occupies within this superfamily of
enzymes. OFORs are important for their ability to generate and use low-potential electrons to
perform difficult chemistry such as CO 2 reduction and so have been targeted by antibiotics such
as metronidazole, which require reduction at potentials lower for activation than those found in
mammalian systems-the NAD+:NADH couple is at -320 mV whereas metronidazole is reduced
at -415 mV (1). The reactions performed by OFORs, with the exception of indolepyruvate
oxidoreductase (IOR), generate electrons with potentials around -500 mV (Table 4.1) and are
capable of reducing metronidazole. OFORs are also known to provide these low-potential reducing
equivalents to other processes, including the reduction of protons, dinitrogen, and sulfate (2-4), as
well as the metabolism of aromatic compounds (5). In the case of OOR, the ability to generate lowpotential electrons enables Moorella thermoaceticato produce carbon-based cellular components,
as well as the cellular energy currency, ATP, from oxalate alone (Figure 1.1) (6, 7).
Table 4.1.
OFOR-catalyzed reaction
Calculateda E'
Oxalate + 2 Fd, := 2 CO2 + 2 Fdred
-492 t 67 mV
Pyruvate + CoA + 2 Fdx acetyl-CoA + CO 2 + 2 Fdred
-515 33 mV
Ketoisovalerate + CoA + 2 Fd,,x isobutyryl-CoA + CO 2 + 2 Fdred
-460 34 mV
a-Ketoglutarate + CoA + 2 Fd,,x succinyl-CoA + CO 2 + 2 Fdred
-480 39 mV
Indolepyruvate + CoA + 2 Fdx S-2-(indol-3-yl)acetyl-CoA + CO 2 + 2 Fdred
-370 37 mV
Phenylglyoxalate + CoA + 2 Fd,, = benzoyl-CoA + CO 2 + 2 Fdred
-460 t 34 mV
aCalculations performed with eQuilibrator 2.0, found at http://equilibrator2.milolab.webfactional.com/. eQuilibrator
2.0 uses estimated thermodynamic parameters to calculate energies for biochemical reactions. Here, the calculations
assumed the pH = 7.0 and the ionic strength = 0.25 M.
108
In addition to dealing in low-potential electrons, OFORs perform key metabolic reactions.
Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the essential transformation of acetylCoA to pyruvate, a reaction that is required by all organisms utilizing the reductive citric-acid
cycle (8, 9), the Wood-Ljungdahl pathway (10), and the dicarboxylate/4-hydroxybutyrate cycle
(11).
2-ketoisovalerate oxidoreductase (VOR), 2-oxoglutarate oxidoreductase (KOR), and
indolepyruvate oxidoreductase (IOR) have all been identified as playing important roles in amino
acid metabolism, degrading small and aliphatic amino acids, glutamate, and aromatic amino acids
respectively (12). OOR may likewise play a role in metabolite degradation, as oxalate is a product
of a number of processes including the metabolism of carbohydrates and vitamin C (13, 14). In
this way, OOR is serving M thermoaceticaby reclaiming carbon and energy from a molecule that
is commonly a waste product.
However, the primary role of OOR seems to be allowing M thermoaceticato thrive on a single
substrate, oxalate, as mentioned above. In particular, the Wood-Ljungdahl pathway appears to be
up-regulated in the presence of oxalate, even under conditions when it is usually down-regulated,
such as when nitrate is available as an electron acceptor (15). OOR is therefore positioned in
a special place in cellular metabolism, with its primary function being the assimilation of an
exogenous nutrient, rather than conversion and recycling of cellular metabolites, as is the case for
the other OFORs.
OOR also performs unique chemistry among OFORs. As discussed in Chapters 2 and 3, oxalate
is a dicarboxylic acid and not a standard 2-oxoacid. The pKas for its first and second proton are
1.25 and 3.81 respectively (16), and so it will be twice deprotonated at physiological pH, and thus
carry a double negative charge. This charge state is in contrast with the other OFOR substrates,
2-oxoacids, which are deprotonated at physiological pH and carry only one negative charge. These
are subtle but crucial differences which make oxalate a much more difficult target for nucleophilic
attack by the TPP cofactor found in OFORs. These differences can best be seen when comparing
resonance structures of oxalate with those of a generic 2-oxoacid (Figure 4.1). Considering just
C2 of the acid, oxalate has three resonance structures, two of which are degenerate and provide
109
a
-oopC0-OO
4~
O 0-0
40 - 00C
- -00C
(+
-
0-
c
0
0
0
0
0
.0
0
R
-
o
0-
b
6-
0
01- 00C -4-)
-00,
R
R
Figure 4.1. The resonance structures of oxalate and a generic 2-oxoacid reveal fundamental differences in
the electronegativity of the C2 carbon. a) Resonance structures of the C2 carbon in oxalate, with carbons
1 and 2 labeled in the leftmost structure. b) Resonance structures of the C2 carbon of a generic 2-oxoacid,
with carbons 1 and 2 labeled in the leftmost structure. c) A combined view of these structures shows
that the negative charge is distributed over all the atoms of oxalate, whereas in a 2-oxoacid, the carbonyl
experiences a partial positive charge (8+) at C2 and a partial negative charge (6-) at the carbonyl oxygen.
C2 with a complete octet in its valence shell, whereas the third is a charge-separate structure.
Because this split-charge state involves two negative charges spatially adjacent to each other,
charge-charge repulsion makes this a much higher-energy state. The C2 of 2-oxoacids, however,
has two resonance structures: a ground state structure with a C-O double bond and a higherenergy split-charge structure, although thi state will not be as high-energy as thc split-charge state
of oxalate. Taking these resonance structures into account, C2 in oxalate should have a partial
negative charge, shared with the entire acid group, whereas the C2 of a 2-oxoacid will have a
partial positive charge.
OOR is unique with respect to other oxalate-metabolizing enzymes in its ability to activate this
relatively electronegative molecule. Aerobic oxalate-metabolizing enzymes, oxalate oxidases and
oxalate decarboxylases, rely on the oxidizing power of dioxygen together with oxalate-binding to a
Mn ion to generate an oxalate-based radical, which causes quick dissociation of the C-C bond (17).
Oxalyl-CoA decarboxylase, the only other anaerobic oxalate-metabolizing enzyme, requires first
that oxalate be activated with CoA. The resulting thioester behaves much more like a 2-oxoacid
and is a better target for nucleophilic attack (18). OOR operates in the absence of dioxygen and
110
CoA. Rather, it makes use of active site residues to stabilize the higher-energy resonance structure
of oxalate (Figure 4. la) and thus make for a more electrophilic C2, amenable to nucleophilic attack
by TPP.
The ability of OOR to activate oxalate for degradation in the absence of other cofactors suggests
that there is still much to learn about the chemistry and function of OFORs. This chapter explores
how OOR fits into the context of the larger superfamily, so that we can better understand how the
functionality of these enzymes relate to their evolutionary history.
Methods
Sequence alignments andphylogenetic tree construction
Sequences of OFORs from various organisms were obtained from the UniRef9O database (19),
with preference for OFORs that have been characterized biochemically. UniProt accession codes
for the 54 sequences are shown in are shown in Table 4.2. Because OFORs lack a ferredoxin
domain (6-subunit), only the a-,
P-,
and y- subunit sequences were used in the alignment. The
alignment was done using the PROMALS3D (20) server (21) with the structures of PFOR from
Desulfovibrio africanus (Da PFOR) (PDB 2C42) and OOR from Moorella thermoacetica (Mt
OOR) as secondary structure references. Alignment parameters are shown in Table 4.3. Alignments
for domains I and VI are shown in Appendix A of this chapter. After alignment, sequences were
trimmed around conserved blocks of residues using Gblocks (22). Similarity matrices were used to
identify sequence conservation blocks with the following parameters: 29 sequences were required
for a conserved position, 45 sequences for a flanking position, a maximum of 8 contiguous nonconserved positions, a minimum block length of 10 residues, and no allowed gap positions.
Trimming resulted in 117 conserved positions from subunit a, and 39 conserved positions from
subunit P. No conserved blocks of sufficient length were identified in the 'y subunit. Using OOR
numbering, the following blocks were conserved: residues 9-20, 23-32, 59-109, 135-155, and
261-283 of chain a; residues 109-128, 134-152 of chain P. These conserved blocks were stitched
together into a single sequence alignment, from which a maximum likelihood phylogenetic tree
was calculated using PhyML (23, 24) using the LG model for amino acid substitution (25), the best
111
Table 4.2. Sequences used in construction of OFOR phylogeny.
Number
Organism
a Subunit
y Subunit
P Subunit
Reference
1
2
3
4
Q8L3B1
Q2W6P3
KONNS5
005651
M3RELO
Q3Z815
Q8L3B3
Q2W6P5
KONQ78
005650
M3NSB I
Q3Z817
Q8L3A9
Q2W6P1
KONTD1
(39)
5
6
Azoarcus evansii
Magnetospirillummagneticum
Desulfobacula toluolica
Thermotoga maritime
Helicobacterpylori
Dehalococcoidesmccartyi
Q56317
M3RIC4
Q3Z814
(40)
(41)
7
Methanosarcinabarkeri
P80521
P80523
P80522
(42)
8
Methanothermobactermarburgensis
P80900
P80902
P80901
(43)
9
Thermococcus guaymasensis
W8CQR1
W8CQB1
W8CQB2
(44)
10
11
Trichomonas vaginalis
Desulfovibrio africanus
Q4KY23
P94692
Q4KY23
P94692
Q4KY23
P94692
(45)
(46)
12
Moorella thermoacetica
Q2RMD6
Q2RMD6
Q2RMD6
(47)
13
Entamoeba histolytica
C4LTX6
C4LTX6
C4LTX6
(48)
14
Cryptosporidiumparvum
Q968X7
Q968X7
Q968X7
(49)
15
16
17
18
19
20
21
Euglena gracilis
Chlamydomonasreinhardtii
Rhodospirillum rubrum
Klebsiellapneumoniae
Giardiaintestinalis
Fervidicoccusfontis
Rubrobacterxylanphilus
Q941N5
L8B958
Q53046
B5XPH3
Q24982
IOA IR4
QlAXJ1
Q941N5
L8B958
Q53046
B5XPH3
Q941N5
L8B958
Q53046
B5XPH3
(50)
(51)
(52)
(53)
Q24982
I0AlR3
Q1AXJ2
Q24982
I0A1R5
(54)
22
Vulcanisaeta distribute
ElQUR8
ElQUR7
ElQUR9
23
Metallosphaerasedula
A4YGO9
A4YG1O
A4YGO8
24
25
Sulfolobus acidocaldarius
Sulfolobus islandicus
V9S813
V9S5T5
VqS5K6
C3N1V3
C3N1V4
C3N1V2
26
Thermosinus carboxidivorans
AlHPK4
AlHPK2
A1HPK5
27
Anaerobaculum mobile
I4BXJ6
I4BXJ7
I4BXJ5
28
Clostridium carboxidovorans
C6PYJ6
C6PYJ8
C6PYJ5
29
Moorella thermoacetica
Q2RI41
Q2RI40
Q2RI42
30
Thermosediminibacteroceani
D9S1F2
D9S1F3
D9S1F1
31
32
33
Sulfurovum sp. AR
Aquifex aeolicus
Leptospirillumferrooxidans
12K9Y4
067229
IOIRW2
12K9Y6
067231
IOIRWO
12K9Y5
067230
IOIRWI
34
Leptospirillumferriphilum
J9ZG44
J9ZD37
J9ZEK4
35
Hydrogenobacterthermophiles
D3DJK1
P84820
D3DJJ9
H3ZL62
(55)
(25)
(43)
Q1AXJO
(6)
36
Thermococcus litoralis
D3DJKO
H3ZL63
37
Methanothermobactermarburgensis
P80908
P80907
P80907
38
Thermosinus carboxydivorans
A1HTT7
A1HTT9
A1HTT8
39
Halobacteriumsalinarum
BOR3GO
BOR3GO
BOR3F9
(56)
40
Thermococcus litoralis
H3ZPH2
H3ZPH3
H3ZPH1
(57)
112
Table 4.2. Sequences used in construction of OFOR phylogeny (continued).
Number
41
42
43
44
45
46
47
48
49
50
51
52
53
54
Organism
Koribacterversatilis
Desulfobulbuspropionicus
Methanothermobactermarburgensis
Helicobacterpylori
Mycobacterium tuberculosis
Staphylococcus pettenkoferi
Thauera aromatica
Halobacteriumsalinarum
Hydrogenobacterthermophilus
Thermococcus kodakaraensis
Thermofilum pendens
Methanolobus psychrophilus
Methanothermobactermarburgensis
Sulfolobus sp.
a Subunit
Q1IQN9
E8RJ94
P80904
068228
053182
HODIR4
087870
y Subunit
Q1IQP1
E8RJ92
P80906
068230
053182
HODIR4
087870
P Subunit
Q1IQPO
BOR4X6
D3DI99
Reference
E8RJ93
P80905
068229
053181
HODIR3
(43)
(58)
(26)
Q8RJQ9
(5)
BOR4X6
BOR4X5
(56)
D3DI99
D3DI98
(59)
007835
007836
007835
A1RYA3
A1RYA4
A1RYA3
K4MML7
K4MBD5
K4MML7
P80910
P72578
P80911
P80910
(43)
P72578
P72579
(60)
Table 4.3. Sequence alignment parameters used PROMALS3D.
Alignment parameters:
Identity threshold above which fast alignment is applied:
Weight for constraints derived from sequences:
Weight for constraints derived from homologs with structures:
Weight for constraints derived from input structures:
Parameters for profile-profile comparison:
Weight for amino acid scores:
Weight for predicted secondary structure scores:
Parameters for deriving sequence profiles from PSI-BLAST searches:
PSI-BLAST iteration number:
PSI-BLAST e-value inclusion threshold:
Identity cutoff below which distant homologs are removed:
Maximum number of homologs kept for PSI-BLAST alignment:
Parameters for detecting and using homologs with 3D structures (homolog3d):
PSI-BLAST e-value cutoff against structural database:
Identity cutoff below which 3D structures are not used:
Align homologs with 3D structures by programs:
Realign target-homolog3d using profile-profile alignment:
Parameters for pairwise alignments between input 3D structures:
Align input structures by programs:
Parameters for aligning sequences within groups in the first alignment stage:
Align sequences within groups in the first alignment stage by:
0.6
1
1.5
1.5
0.8
0.2
3
0.001
0.25
300
0.001
0.2
fast; tmalign
yes
fast, tmalign
mafft
113
-
=111=---IT.-
a
N6
7
3
9
Figure 4.2. Phylogenetic and sequence analysis of OFORs. a) Maximum likelihood tree of 54 OFORs.
Bootstrap values for nodes of 90 or greater are indicated in parentheses, and a scale bar showing evolutionary
distance is at the bottom left. Numbers by the name of each genus refer to the protein sequence data in
Table 4.2. An asterisk next to a name indicates that a protein has been biochemically characterized. All
monophyletic groups, with the exception of Group 4, which is paraphyletic, are validated with bootstrap
values of 90 or greater, and are indicated by colored regions and numbers 1-9. b) The groups in a) are
identified by their putative substrate, along with representative sequences for functional sites in the enzyme
active site. c) Stereoview of functional sites from b) are highlighted in pink in the OOR active site, shown
in stereo. d) Stereoview of functional sites from b) are highlighted in pink in the PFOR (PDB 2C42) active
site, as in c).
114
b) Monophyletic* groups with putative substrates and functional sites.
Group
3
Substrate
Chains
Site 1
Site 2
Site 3
Site 4
Site 5
Site 6
Oxalate
afry
YPIRP
R
DPP-GDF
N
R
Oxalate
Phenylglyoxalate
aNy
YPIRP
R
DDP-GAY
N
Q
Q
apys
YPITP
R
ITP-QCV
N
T/G
R
apys
YPITP
R
SAP-INI
N
L/F
R
a
apyS
YPITP
R
STHALSI
N
A/S
K
YPITP
R
NAP-LSI
N
-
K
ap
apya
YPGTP
YPITP
A
R
PSM-FSS
PGLGNIG
M
M
-
Q
-
Q/E
YPITP
R
PSTGLPT
M/L
P/F
I
Pyruvate
6
7
Indolepyruvate
Ketoisovalerate
a-ketoglutarate
ap;
apy8
R
*Group 4 is paraphyletic
c
2
2
1
5
4
OOR
6
3
1
5
4
OOR
6
V
d
5
PFOR
5
PFOR
115
tree topology from both NNI (nearest neighbor interchange) and SPR (subtree-pruning-regrafting)
searches, and 100 bootstrap calculations.
Results
The majority of the 54 enzymes considered grouped into eight distinct monophyletic clades
(a monophyletic clade is a group that shares a common ancestor), supported by bootstrap values
greater than 90, which is generally considered a good support, with values of 95 and higher offering
very good support (Figure 4.2a). Group 1 contains Mt OOR along with other primarily bacterial
enzymes and so was assigned oxalate as the putative substrate (Figure 4.2b). Group 2 contains
mostly archaeal enzymes, none of which have been characterized. Group 3 contains putative
NAD+-dependent phenylglyoxalate dehydrogenases. Group 5 contains full-length fusion PFORs
from bacteria and eukaryotes. Group 6 contains putative bacterial pyruvate oxidoreductases (PORs,
to distinguish from the fusion proteins in Group 5). Group 7 contains archaeal indolepyruvate
oxidoreductases (IORs). Group 8 contains two putative ketoisovalerate oxidoreductases (VORs).
Group 9 contains 2-oxoglutarate oxidoreductases (KORs).
Of the seven enzymes that formed a paraphyletic group, i.e. a group with all descendants of
the last common ancestor, (Group 4), six have been biochemically characterized. Five of these
enzymes were characterized as PORs, and one, enzyme 36 from Thermococcus litoralis was
characterized as a VOR (26).
Discussion
The tree shown in Figure 4.2a is consistent with other trees previously calculated for the
OFORs and PFORs (26, 27). For the most part, previously identified and characterized enzymes
cluster together according to their identities. Full-length PFORs (Group 5), IORs (Group 7), and
KORs (Group 9) all cluster together into well-defined monophyletic groups. VORs are notably
polyphyletic, with some of the enzymes being related to KORs (Group 8) and others related to
PORs (enzyme 36), which suggests that VORs may have arisen.at two separate times in evolutionary
history. PORs (Group 4) are paraphyletic, which may indicate that either not enough sequences
116
were used to determine the relationships of these enzymes, or it may be indicative of greater
functional capacity than previously thought for this family of enzymes.
Consistent with the existence of a broad functional diversity in OFORs is Mt OOR, which
performs unprecedented oxalate oxidation chemistry. As shown in the previous chapters, OOR
is able to perform its chemistry by reorganizing the active site after oxalate binding, enabling the
enzyme to change the charge landscape of the active site during catalysis (Chapter 3). Many of
these residues are located in specific functionality sites and can be compared to the structure of Da
PFOR as well as the aligned sequences of these other OFORs (Figure 4.2b-d). Group 1 consists of
enzymes that are nearly identical to OOR in all of the relevant catalytic positions, including site 1,
which provides Arg3 1 a as part of a YPIRP motif in OOR to stabilize the additional negative charge
of oxalate, and site 3, the Switch loop containing Asp 116a in a DPP-GDF motif, (see Chapter 3),
which appears to be playing a role in driving oxalate oxidation by OOR. The presence of all of
these conserved motifs in Group 1 enzymes suggests that all of these enzymes are OORs.
Like Group 1 OORs, Group 2 enzymes also contain the rare YPIRP motif that places Arg3 1 a
in the active site. However, these enzymes have a different Switch loop, with a critical Asp--Ala
substitution in place of Asp 1 6a of OOR, which was proposed to help drive oxalate oxidation
in OOR. Without biochemical data, it is impossible to say exactly what this mutation means for
the chemistry of this enzyme clade, but a few contextual observations can be made. An OFOR
from Sulfolobus solfataricus (Uniprot Q7LX68, Q7LX69, Q7LX70), which has 96% identity to
subunit a of enzyme 25 in Group 2, was reported to be tightly regulated by a Leucine-responsive
Regulatory Protein (28). It was determined through gene disruptions that, unlike other OFORs, this
enzyme is likely not involved in central metabolism. It was also noted that immediately upstream
on the same operon of this OFOR is a transport permease that has homology to oxalate/formate
antiporters. Therefore, the circumstances suggest that this OFOR from S. solfataricus, and by
extension all of the Group 2 enzymes, are OORs. Although the lack of conservation of the Switch
loop is concerning, it could be that the presence of the YPIRP motif is enough to classify these
enzymes as OORs.
117
An intriguing observation of the putative Group 2 OORs, assuming this assignment is correct,
is that they are primarily from metal-leaching archaea (29-31). Although these organisms are
believed to leach metals primarily through the oxidation of elemental sulfur to sulfuric acid, it
is also known that oxalate can serve to leach metals (32). An intriguing possibility is that these
organisms are using OOR to produce oxalate through CO 2 reduction to aid in the retrieval of metal
ions from ores. Alternatively, OORs may be produced as a way to take advantage of metals leached
by heterologous oxalate, similar to how organisms compete with and for siderophores (33). Either
way, it will be interesting to gain a better understanding of the role of OORs in nature by studying
them in a wide variety of environments.
Looking at some of the other functional sites (Figure 4.2b), there are a few interesting features
in the more highly conserved sites. Site 2, which corresponds to Arg109a in OOR is strictly
conserved across all OFORs with the exception of IORs, which have an alanine in that position.
This Arg residue is believed to be important for binding the acid group of the 2-oxoacids, so it
will be interesting to see whether IORs have made up for the functionality of this site with a
corresponding mutation at another site. Site 4 is also strictly conserved as an Asn in Groups 1-6.
This Asn residue sits right along the electron transfer pathway between TPP and the proximal
[4Fe-4S1 cluster in both Mt OOR and Da PFOR, and may be making important interactions with
both of these cofactors, as well as with bound substrate (see Chapters 2 and 3). In Groups 7-9,
however, this site is mutated to a Met or Leu residue. Interestingly, an enzyme from Sulfolobus sp.
strain 7 (#54 in Group 9), which was demonstrated to have both POR and KOR capabilities, was
shown to require Leu in site 4 for POR activity, whereas a mutation to Asn was still capable of
KOR functionality (34). These seemingly contradictory results will most likely only be answered
once the structure of a KOR family member is determined.
Site 6 is a functional site that has not been explored biochemically to the best of our knowledge.
It is of interest for its close proximity to the proximal [4Fe-4S] cluster as well as the probable
hydrogen-bonding interaction with site 4. It would appear that both of these interactions are once
again conserved in Groups 1-6, whereas it is again altered in Groups 7-9. It may be telling that
118
in most instances in which site 4 is a hydrophobic residue, site 6 is either hydrophobic (Group 9)
or carrying no charge (Group 7). The exception to this trend is enzyme #38 from Thermosinus
carboxidivorans,a putative VOR. Given the conservation of these residues and their key placement
adjacent to the electron-transfer pathway, it is likely that they are playing a role in tuning the redox
potentials of TPP, the proximal cluster, or both.
The final two sites in this discussion are sites 3 and 5. These sites were chosen based on their
involvement with the active site in Mt OOR. As already mentioned, site 3 is the Switch loop in
OOR that is capable of drastically altering the charge landscape of the active site, a feature that
is proposed to drive oxalate oxidation (Chapter 3). It has already been mentioned that this feature
is not strictly conserved in putative GORs. The role that the Switch loop plays in forming the
boundary of the substrate binding pocket along with its lack of strict conservation suggests that it
may play a larger role in fine-tuning substrate specificity, although more work will need to be done
to ascertain exactly how this role is manifested across the other groups.
Site 5 is Gln2 11 a' in OOR that interacts with bound substrate through an ordered water
molecule and is proposed to facilitate the donation of an extra hydrogen bond to oxalate prior
to nucleophilic attack by TPP, as well as in the final stages of catalysis to lower the pKa of a
bound reaction intermediate (Chapter 3). This Gln residue is only present in clades that have been
assigned as putative OORs (Groups 1 and 2), consistent with both the assignment of these clades
as well as the function of this residue in binding oxalate. Unlike site 3, however, it is not clear that
site 5 will have any functional relevance outside of GORs.
One feature of OOR that was not found at all outside of Group 1, and therefore could not be
assigned as a "site", is the domain III plug loop. This 13-residue insert into domain III effectively
plugs a solvent channel to the active site, but it becomes disordered when domain III rotates
away from the active site (Chapter 3). This insert was initially noted because it occupies the same
channel that is occupied by the C-terminal domain VII of D. africanus PFOR (enzyme 11 in
Group 5), although this same domain VII is missing in other PFORs. However, it is has become
evident that the OOR plug loop plays a larger role in catalysis by providing interactions to the
119
substrate-binding residues of the active site. Domain III is one of the least conserved domains of
this superfamily of enzymes, a fact that made it impractical for use in phylogeny construction.
It would be interesting to see, when other structures of OFORs are determined, if there are other
insertions into domain III that play a similar role to OOR's plug loop. Both Da PFOR and Mt OOR
use non-conserved features to regulate access of substrates into their enzyme active sites, raising
the question of substrate access for other OFORs. There are most likely still plenty of surprises in
store from OFORs.
Conclusion
The work presented in this thesis has provided structural insight into the chemistry of oxalate
oxidoreductase from Moorella thermoacetica. Three X-ray crystal structures of Mt OOR have
provided intimate snapshots of OOR prior to oxalate binding and at discreet steps along the reaction
pathway. These structures revealed two covalent adducts to TPP that had not previously been
visualized, [oxalate-TPP] and [C0 2 -TPP], as well as how OOR interacts with these adducts. Before
oxalate binds to the active site, the active site is poised with various hydrogen-bond-donating and
positively charged residues to lure the twice negatively charged oxalate into the active site. These
hydrogen-bonding interactions are proposed to stabilize a more electrophilic resonance structure
of oxalate, which is a better target for nucleophilic attack by TPP.
Following formation of the [oxalate-TPP] adduct, domain III is proposed to rotate away from
the active site, causing the plug loop to become disordered and allowing Arg3 1 a, one of the activesite residues unique to OORs, to swing away from the bound intermediate. At the same time, in
a movement that may or may not be correlated with domain III motion, the Switch loop flips
around to the Asp-in conformation, bringing AspI 16a into contact with the intermediate. This
Arg-for-Asp swap may be responsible for destabilizing the [oxalate-TPP] adduct, leading to the
first decarboxylation. It is likely that the Switch loop continues to drive the reaction, leading to
transfer of both electrons into the [4Fe-4S] clusters and decarboxylation of the bound intermediate.
In driving the reaction after the first decarboxylation, the Switch loop would effectively fill in for
CoA, which is required in every other OFOR. Thus in all of these protein-substrate interactions, it
120
is evident that OOR is uniquely tailored among OFORs to activate and catalyze the oxidation of
oxalate.
When considering oxalate metabolism as a whole, we see that OOR in a way completes
the picture. As oxalyl-CoA decarboxylases are the anaerobic counterparts to aerobic oxalate
decarboxylases, so OORs are the anaerobic counterpart to aerobic oxalate oxidases. Unlike these
other pathways, however, OOR is uniquely capable of capturing the electrons released in the
oxidation of oxalate, a feature that allows M. thermoaceticato grow exclusively on oxalate via the
Wood-Ljungdahl pathway. Based on the structural analysis as well as phylogenetic analysis, it is
likely that a number of other organisms also have this capability to metabolism oxalate with OOR.
One open question is whether OOR will turn out to be relevant to human gut microbiome.
Oxalate toxicity is a cause of renal failure, kidney stones, and crystalline arthritis, and it is important
to be able to identify all the potential players in oxalate metabolism in the human gut. To date,
most studies of probiotic-based treatments have focused on Oxalobacterformigenes,which uses
the oxalyl-CoA decarboxylase pathway. Although organisms in which OORs are found are not
commonly associated with the human gut, M thermoacetica has been found in various oral and
stool samples (35). The limited number of complete microbial genomes that have been sequenced
may also make it difficult to identify OOR-utilizing organisms in the microbiome. As of five
years ago, there were complete genomes for only three acetogens, including M thermoacetica.
In general, however, acetogens are known to make up an important part of the gut microbiome,
feeding off H 2 and CO 2 (36), and whether or not OORs will be found in other microbiome-associated
acetogens remains to be seen. With the rapidly expanding technologies that allow sequencing of
more and more microbial species as well as the identification of smaller and smaller populations
within larger microbial communities, it will be interesting to see how our understanding of oxalate
metabolism in the human gut develops.
Apart from the microbiome, there is also interest in using oxalate-metabolizing enzymes to
directly degrade oxalate. These therapies have primarily made use of oxalate decarboxylases, which
may be effective, but must certainly be limited in the human gut by their requirement for dioxygen.
121
Oxalyl-CoA decarboxylase would be ideal for an anaerobic environment, but this enzyme requires
the use of CoA as well as a separate CoA transferase, so it would be impractical for an enzymatic
therapy. OOR, however, may be ideal for enzymatic therapies for treating oxaluria. As an anaerobic
enzyme, it ought to be perfectly capable of functioning within the human gut. Furthermore, it only
requires oxalate as a substrate. Additionally, the high-temperatures and low pHs in which the
thermophiles of Group 2 live suggest that some of these archaeal OORs may be particularly stable
to degradation, which could be an important feature for oral therapies (37, 38).
Whether OOR ends up playing a larger role in oxalate metabolism remains to be seen. What
is certain is that OOR has proven to be an enzyme full of surprises for the enzymatically curious,
intruigue for the evolution-minded, and an all-around positive experience for one small 2-carbon
molecule known as oxalate - that is, until the loop flips.
Acknowledgments
This work was supported in part by National Institutes of Health Grants GM069857 (C. L. D.)
and by the National Science Foundation (NSF) Graduate Research Fellowship under Grant No.
1122374 (M. I. G.). This research was also made possible by the support of the Martin Family
Society of Fellows for Sustainability (M. I. G.). For more information about the fellowship please
visit http://dusp.mit.edu/epp/project/martin-family-society-fellows-sustainability.
C. L. D. is a
Howard Hughes Medical Institute Investigator.
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Appendix A - sequence alignments
Sequence alignment of conserved blocks of domain I from 54 OFORs (numbering of sequences is the
same as in Table 4.2). Residues that are poorly conserved are shaded purple and green; residues with
average conservation are shaded yellow; and residues with good conservation are shaded light pink to red.
Alignment visualized with TCS (61).
1
2
3
4
5
6
7
8
9
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29 (Mt QOR)
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
cons
OOR numbering
6a
23a
---------------------------------------------
LAEGNEAAALGVALARP ----VCDGNEAAAWGACLSRP -----
DM
DH
-------------------------------------------------------------------------------------------------------------------------------------
VCDGNEAAAWGVAKAKP ----- DM
AVTGAEAVANAMRQIEP ---DV
VWDGNTASSNALRQAQI ---DV
GVEVSIALSEAVGLCNA ---DV
VVEGSYAVAHSAKVCRP-----NV
VISANQAVAEAAKLAKP ---KV
-----------------------
VMKANEAAAWAAKLAKP
----
VEHVAKLIAD ------------ GRM
VEYLAQFIAD ------------ GRM
AEYIAQFVAD ------------ GVI
VEYFARFVAD ------------ GVV
VQNYGSFKDN ------------ GYV
VEHLAEMVAD ------------ GEL
VEHLSQFMAD ------------ GEI
SEYLAKYVAD ------------ GEL
PEKISEFVAN ------------ GEL
GELVDAWIAQ ------------ GRGEEADDWAAQ ------------ GRAEIADEWAAH ------------ GRAENADVWSSQ ------------ GRSEVADSWMSR ------------ GRGELADVWMAQ------------GRGEMVDQWASE ------------ GRGELSDEWSAK------------GLAENVDEWAAK------------GKAEMIEQYAAQ------------GKMNALASMIAN------------GEF
KV
PGDGNTAATSVAYQLS ----ET
TTDGNTATAHVAYAMS ----EV
TLDGNTAAAHVAYAMS ----EV
SVDGNQAAAYVSYALS ----- - D'V
IVDGCVAACHIAYACS ------ EV
VDGSQATSVAYALS --- DI
SDMDGNEATALIAYAVS ---- DV
-------------AIDGNEACASVAYRVS ----- EIV
----------------------TMDGNTAAAWISYAFT ---DV
----------------------CIDGATATSMMVYRLI ---DF
----------------------VWNGARAIAEAVKVADV---- Dh
----------------------LTTGTQAVAQAVKLADV---- D%
----------------------VGTGTDAVAYALMLADV---- D%
----------------------LLNGTQAVAYAAMYADV---- D\
----------------------LMN6TQAVAHAAMYADV---- D%
----------------------LMNGTQAVAHAAMYADV---- D%
----------------------RGTGLTAVADAWQLADI---- D%
----------------------LITGVVATAEAVRLADV---- D%
----------------------RMSGCVATANAIKLANV---- D\
----------------------NISGCVAVAHGVRLADV---- D\
----------------------NISGCVAVANGVRLADV---- D\
----------------------FITGAQAMAEAVKRANV---- D'
----------------------FITGSQAFAEAVKRANV---- D\
----------------------FITGSEAAKEAIRRANV---- D\
----------------------FLTGSEVIKEAVKRASV---- DI
----------------------FMTGSEAVKEAVKRASV---- DA
----------------------VVSGNYAAAYAAKHARV---- E\
----------------------MVKGNTAVIIGAMYAGC---- DC
----------------------LMKGNEAIGEAAIVAGC---- Rfr
----------------------LLNGDEAIGMGAIAAGC---- RF
----------------------FIQGDEAIARAAILAGC---- RF
----------------------FLDGDHACCEGALAAGA---- RF
----------------------YITGDVACAEGAIAAGC---- K?
----------------------FIQGNDACARGAISAGC---- RF
----------------------ISDGNELVAKAAIEVGC---- RF
----------------------QISGNTALAYGIVVAGMW
----------------------YMIGNDAIGLGAISAGS---- RI
----------------------SITGNEAAGLGAVRGGV---- RI
----------------------LMSGSHAIAYGAIDAGC---- RI
----------------------IMEGNQAIAKGAVVAGC---- KI
----------------------LLLGNHAIARGALEANI---- A\
----------------------LLLGNEAIARGALEAGI---- S\
----------------------YMLGNDAIARGIVEGGA---- Q\
----------------------FLLGNEAAVRAAIESGV---- G1
----------------------WLDGNTAVAIGKIYGGV---- RI
--------------------------------------------------------------------------------------------------------------inAV
MQAVAKLIAD------------GEL
MQTVAKLIAD------------GYM
MDTISKLIAD------------GQL
MDTISKLIAD------------GEL
MDTISKLIAD------------GEL
MAEVAKRIAN------------GQM
MSELARMIAD------------GEL
MSELSRMVAD------------GEL
MSELARMVAD------------GEL
MSELARMVAD------------GEL
MHLVGDIYAQ------------GHI
MHLVGDLYAQ------------GYV
MQQVGALWAE------------GYV
SSLVGELFAD------------GYV
AHLIGELWVE------------GYV
IEKIAEFIAN------------GEV
LHEASRYFPL------------VGTEYMAKRMPQ------------VGMEYLTGRIEQ------------FGFEAMSLYMPL------------VDVERFAARVPT------------VGAEHMAERLPD------------IGAEEMAVLLPG------------EGMHAMSVALPN------------CGLHELSKHKN-------------FNMEYMIENLPK------------VGLEWLAPNLAK------------LGFTIMTQLLPD------------MGGNYIVEDLIR------------VGTDTMAAVAKK------------AGVETLAEVGER------------YGIGTLASMKER------------D-GNVLSKIAKR------------AGSVYIEAHQDET-
i:*
*
Sequences
Site 1
127
cons
RTSGMRLPIVLTIVTRDGITP-QSVWGGHQDAM
RTPGLRLPMVVSLVTRDAVSP-TCVWGGQQDAM
RTPPLRLPIVMSIVTRDGITP-QCVWGGQQDAM
IAASYRLPIVMPVVNRALSGP-INIHCDHSDAM
QASGMRLPIVLNLVNRALAAP-LNIHGDHSDMY
NTSGMRLPVVMTVANRAVSAP-INIWNDHQDAI
VASSMRLPVIMAVANRALSGP-LSVWGDHSDVM
AAAGLRNPIVMANANRALSAP-LSIWNDQQDSI
IAAGMRLPIVMAIGNRSLSAP-INIWNDWQDSI
KIIGEMCPAVSHISARCIATSALSIYNDHGDIY
KISGELLPGVFHVTARAIAAHALSIFGDHQDIY
KIAGELLPCVFHVAARALSTHALSIFGDHADVM
KIA6EHLPCVFHVTARALAGQALSIFGDHSDVM
'KIAGELWPCVFHVTARAIATSSLSIFGDHNDIM
'KIAGELMPSVIHVAARELAGHALSIFGGHADVM
XIAGELTPCVLV T ARALAKHALSIFGDHQDVM
'KIAGELTPFCMHVTARTLATHALSIFGDQSDVM
KIAGELLPGVFHVSARALATNSLNIFGDHQDVM
'HMAGERIPFVLKVATRTVGMAATSLATDHTDVY
VTALSQLPVVGAIGTRALDDP-GNFGMEWSDVL
VTPSLRLPVVAMIGNRALDDP-GAFGVEHNDAM
'AISTDRYPVVALIGNRALDDP-GAYGVEHNDAL
VTATDRAPVVAVIGNRALDDP-GAYGVEHNDAL
VTATDRAPVVAIIGNRALDDP-GAYGVEHNDAL
VTATDRAPVVAIIGNRALDDP-GAYGVEHNDAL
PIAGGRCPIQALIADRTLDPP-GDFGSEHTDCL
IPISGERFPVQMAIADRTLDPP-GDFGSEHTEAE
PISGERLPLQMAIADRTLDPP-GDFGSEQTDAL
,PISGERLPVQMAIADRTLDPP-GDFGEEHTDAE
'PISGERLPVQMAIADRTLDPP-GDFGSEHTDAE
'SWSGHRVPAVLGILTRVVNAP-LSIQPDNIEMA
'SWPG RIPAVLGVMTRVVNAP-LSIQPDNVEIS
*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29 (Mt OR)
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
51a
kSWPGGRMPIVLLIMCRVINAP-LSIQPDNAELA
'MWAGSRLPIEVVFTVRGVNSP-LSIQPDTLEIA
'MWAGTRIPVQLVLMARGVNAP-LSIQPDNLEVS
IWAAGARLPIVMVDVNRAMAPP-WSVWDD TDSL
FLAGAELPAVIVDVMRAGPGLGNIGP
Q
YIAGAELPCVIVNIMRGGPGLGGIQP
6 Q
iLVATSETPLVIANVMRSGPSTGMPTKQEQGDLN
iYAIMTETPLVLVNVQRSGPSTGQPTLAAQGDLM
iYAVMTETPCVFVNVQRGGPSTGLPTLPAQGDMM
'LAVCTETPCVIVNVQRTGPSTGLPTQTGQGDMM
iYAAMTETPLVIVDVQRGSPSTGQPTMASQSDMM
iYSFMAEIPLVIADVMRSGPSTGMPTRVDQGDVN
iLGVMTELPLLVIDVQRGGPSTGLPTKTEQADLL
iLSGMTETPFVIVNTQRGGPSTGLPTKQEQSDLM
iLAVASETPITIVNVMRGGPSTGIPVKSEQSDLN
iLAEMTETPLVLLEAQRAGPSTGMPTKPEQADLE
iYAGMTELPIVIVDVQRVGPATGMPTKHEQGDLY
.SSVGMGVEGGFVIMVADDPSM-WSSQNEQDTRV
ISLGYTGVVGGLVIVTADDPNAHSSQNEQDNRI
ITLAYTGIKGSMVIIVADDPSC-HSSQNEQDTRR
iSVAYTGVRAGMVVLSADDPSM- FSSQNEQDNRH
iWAGMNEVPVVITYYIRGGPSTGLPTRTAQSDLI
*
Sequences
Site 2
128
Site 3
IMAFKIAEHHDVMLPVNVCLDGNYLSYGASRVELPDQAVVDE ---- FMGEKNVN--'IAYKVAEHPDVMLPVNVNHDGNYLSYGVARVELPDQSVVDA ---- FLGEKQVN--[LAYRIAENRDVMLPVNVCHDGNYQSYGVEQVLLPDQDMVDG ---- FLGEKNTN--[LAVRLAEHEDVRLPVMVNLDGFILSHGVEPVEFYPDELVKK ---- FVGELKPM--.MAFRIAEHQKVRVPTIVNQDGFLCSHTVQNVRPLSDAVAYQ---- FVGEYQTK--[CSFKIGEDKRVLLPVMVHLDGFHLSHVIEPIDMPERAQVDA ---- FLSVNDY ---FLPPFQP--->QLYKIAEDNEIMVPGMVCMDGFILSHVYEPVVLLEQDLTDN ---LLSYRVSEDRDVLLPSMVCLDGFILTHTVEPVDIPSQDEVDT ---- FLPEFQPQ -LIAYKVAEDERVLLPAMVGFDAFILTHTVEPVEIPDQELVDE ---- FLGEYEPK -WVAHLTTIHTG--LPFIHTFDGFRTSHEINTYEELDNETLWP ---- LIDQKAL
LVAHLAAIESN--VPFMHFFDGFRTSHEIQKIEVLDYADMAS---- LVNQKALA
LVAHLATLKAR--VPFVHFFDGFRTSHEVQKIDVIEYEDMAK---- LVDWDAIR
[VAHLATLEAS--LPFMHFFDGFRTSHEIQKIHQLTYDQIKS---- LVDMKYVD
LVSHVSTFECS--VPFVNFFDGFRTSHELQKIDMISYETIKK ---- IFPYEKLIK
LISHVATLKSS--IPFVHFFDGFRTSHEVNKIKMLPYAELKK ---- LVPPGTM
1
QDAIK
LVSHLATLRAS--VPFVHFFDGFRTSHEINTIHLIDNEAIKS
SVAHGASLESR--IPFLHFFDGFRTSHEVNKIELMTDDDLHA ---- MIDDDLV
WVAHLAAIAGR--IPFINFFDGFRTSHEIQKIEVLAYEQLAT---- LLDRPALE
LVAHTTATNVQ--AAFCHFFEGYRVSHQFETIQVFKDDADIK --IFLSEDQLA -LVAYRVAEDKRVLLPHFVVLDGAALTHVATPIVPVTKEQATD---- FLPPYKPP -LMAYRVGEDPRVSLPVGLGVDGAFITHSQTLIKIPDQETVNE ---- FLPPYDLG--LIAYRVAEDPRVMLPVGISMDGAFLTHSEHVFRIPPKSKVQK ---- FLPPYDLG--LLAYRIGEDPRVLIPVGVSMDGGFLTHSEQIVRLPDKELVKN ---- FLPPYDRG--LIAYRVGEDPRVLLPVGVSMDGGFLTHAEQIVRLPPKELAKE ---- FLPPYNRG --LIAYRVAEDQRVLLPLGISMDGGFLTHSEQIVRLPPKELVKN ---- FLPPYNRG--LMYFRIGEDPEIMLPQIVAMDGYFVTHITQEYEMPDQEQVDK ---- FLPPFKPQ--LLYYRIGEDPRVLLPQYACQDGYFVSHIAGEVEIPDEGQVKE ---- FLPPYKNK--LMYYRIGEDPRVLLPQYACQDGYFVSHILGEVDVPSQEQVDS ---- FLPPYKNH -LIYYRVGEDQRVLLPQYACLDGYFVSHILGPVDIPDEAQVKE ---- FLPPYKNH -LIFYRVGEDPRVLLPQFACLDGYFVSHILGPVDIPEPEQVKE ---- FLPPYKNH --
LAAFAITEKVDVYIPVAVATEGFFVTHAKGYVDMTADDLCLN ---- DFDPVAAP -LAGFVISEKVDVYLPVVVATEGFFVTHAKGYVELPPEDMKLP --- PRDPYKA- --LKAFIISERPEVTLPVAVAVDGFFVTHARGYVMMPSKDIKLP---- PRDPYHEA--LKGYIVAEKPEVHLPIGVIVDGFFVTHTKDMVMMTPENRTLP --- HYNPYASP -PYNPYRAP -LAGFVVAEQPDVHVPVITVVDGFFVSHTRETVMLPPDDIALP --LMAFKIGEHEKVNLPVMVVESAFILSHTYDVVDMPEQEEIDE---- FLPPRKPLMDAFELADKYR--NPVIILADAV-LGQMAEPLRFPERAVEHR----
TVREAG-----WIQVYCESVQE
TVKEVG-----WIHMYCETQQE
TVKEVG-----WIQMYCETNQE
AERDSG-----WIQLFAETNQE
LSRDSG-----WISLCTCNPQE
SCRDIG-----WIQIFTENGQE
AQRDTG-----WMQLYVEDVQI
AERDSG-----WMQIYAESGQI
SERDTG-----WLQFYAENNQI
ACRATG-----MPILFSNGVQf
AARQTG-----FAMLASSSVQE
AARQTG-----FAMLSSASVQI
ACRSTG-----FCLLSSHNVQ(
AARQTG-----WAFLGAMTWit
AVRQTG-----WAMLCSHW
AVRQTG-----WAMLCSHSVQI
ACRQTG-----FAILASASVQI
AVRQTG-----CAMLVENNV
AIEGTN-----MCALS$CP
MFRDYG-----WLISWAKTVQI
AVRDLG-----WLLFWVEDAQI
MVRDVG-----WLLVWVDTAQI
MVRDVG-----WLLVWVDTAQI
MVRDVG-----WLLVWVDTAQI
MVRDVG-----WLLVWVDTAQI
TLRDNG-----WIYGWAQDAQI
SCRDQC-----WIQGWAATPQI
CCRDQG-----WIQGWASTPQI
CCRDQG-----WIQGWASTPQ
CCRDQG-----WIQGWASTRQI
YMMHCG ----- AVMLHAENQQ
YLLNSG ----- LVVLHAENQQ
YLINTG-----NIVFHAENQQI
FMLETG-----MLLWHAETAQ
FLLDTG-----CMIWYAETAQ
AQRDTG-----WLQFYAENNQ
LVKGGG NYRNIVLAPNSVQI
ATKGGGOGDYHNIVLAPNSVQI
MMLKGGHG IPRFVVAPTSVA
SLIVLTPATVI
QAIWG
QARWGSWDYEIIALCPNSPQI
QARWGS DYELIALAPSSPQI
QARWGSHGDYEIIALSPSSVQ
FLRHPIHGDFKAVALAPASLE,
QALYGRNGESPVAVLAPRSPA
QMIYGTHGDIPKIVVAPTDAE
IALYGMHGDAPHLVVAPNSLA
HVLYTSQGDSHRVAFGPKDPK
HAIYSGHGEIPRAVLAPTNVE
YAKFAN- VPVLEPSSPH
IPVFEPSSPQ
YGLHSYIPCLDPSTPQ
YSQFAL VPLLEPSNPQ
YARLAW FPIFAGHGEFPKIVLASGDHA
ILAFDLADQYR--NPVMILGDGA-LGQMMEPVEFT
-- -
VEAFNLAEKYQ--LPVYVTADLS-AVTERT--IRAfNLAEKYR--TPVILLTDAE-VGHMRERVYAPHPDELEL -- IYR
EPR
IKAFNYAEKYR--VPALVMLDEV-VGHMTEKVVIPTADQIEV-IRAFNLSEKYR--VPVLLMADEI-VGHMSEKVVIPEASKIRL --- IDRPKPTG
VRAFNLAEEYR--VPVVVLSDEI-VGHMREKITIPDKVEIR---
-
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29 (Mt OR)
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
132a
---
Sequences
1
ARAFNLAEMLM--TPVFLLMDET-VGHMYGKVQIPDLEEVQK-- TTINR
LEAVRIAVSYH--TPVILLSDGA-IANGSEPWRIPDVNALPP-- IKH
RGEL
MEAFNLAEIYQ- -CPVIILSDLQ- LSLGKQTVEQLDYNAIDI
QWAVHLADTLQ- -TAAIVLSDQS- LGQSRATIAPPADPGLRRTAFEIAYDYQ- -IPVILLYDQK-LSGEYRNVDASF
IN
VEAFNLAEKYQ- -IPVIVLTDAS-LSLRAEAFPTPKVKDIKV
KYAfELSEKFK- -HFVILRTTTRSSHARGDVGLE------RDLYDLSEKYS- -TAVFLRTTTR-LSHSRGEVTLGELRGAG
SYASELSGKMQ- -MPVIFRPTTR-ISHGKSDVVLGNVTQNK ------NHAFELSEEYR- -IPVLLRTTTR-VSHMRGVVEAGERRAE
iv
I
-IWALNLAEKYQ- -TPVIHLVEKT-LANSYSTIPYEELELD
cons
129
Sequence alignment of conserved blocks of domain VI from 54 OFORs (numbering of sequences is the
same as in Table 4.2). Residues that are poorly conserved are shaded purple and green; residues with
average conservation are shaded yellow; and residues with good conservation are shaded light pink to red.
Alignment visualized with TCS (61).
1
2
3FLKNNE
4-
12P
numbering
SqecsOOR
HITTAI ----------------------------------------------- DWEDLFAPGLA6CQ6CNTELLMRHTLRRVG
FRKAGS ----------------------------------------------- AKEDLFVPGLS6CQGCNSELIMRHAMRKIG
-------- M--------------------------------------DKEDLVAPGISSCLGCNTELVFRHTLRKVG
EOKEIGITQGHRLCPGCGAPTKVIA
----------------------------
5
---- Ii----------------------------------------QSAEKFQGSHLLCPGCG6i~klIVREVLNAVD
20
---- I----ISLREI -------------------------------
28
---- f------- KSIKQA ---------------------------------- PVEEYYVPGHRTCACGALQRLVAKAAG
pLTYRLVAKAAG
30
--
31
- ---
LL---------------------------------- PKRELFAPGHRGCIGCGEALAVRLATKAMD
- ---6--KTYITSGHSGCAGCCD3AFAAKFTLMGAG
.................
--------7
8-------------------------------------------------------------PEEEFLAPGHRGCAGCGATVGVRLALKVLG
TTREYWAPGHAACAGCGCAZALKLATKAFS
-----------------------9
GK- -----ENRGLLPPTNVRNVQFRQPLIEFNGACQGCGETAICKLLTQLYG
- LSMT ---------- YRNAKLEEE
10
ALVMQ ---------- PLDTQRDAQVPNLEYAARIP--- VKSEVLPRDSLKGSQFQEPLMEFSGACCG6ETPYVRVITQLFG
11
ALTMV ---------- PLEEVTAVEEANYNFAEQLP --- EVKVNFNPATVKGSQFRQPLLEFSGACAGCGETPYVKLVTQLFG
12
- LSMT ---------- PFEQVSEVESKNWEFAMTLS --- PKNSLSDRSNIKTTMIHQPYLEFSGACE4CRETALVKLITQLYG
13
-LHME ---------- GLQKMEAVEKTHWDYLIGLP --- NKAEKFDRTTVKGSQFQQPLLEFSAACEGCGE1'PYVKLLTQLFG
14
-LEMT ---------- DAFTATPVQRTNWEFAIKVP--- NRGTMTDRYSLKGSQFQQPLLEFSGACEGCETtPYVKLLTQLFG
15
LQSV ---------- PINSVLEVETANWDFTGTLP--- ARTDIMDKATVKGSQLQPPLMEFSGACEGCEPYVKLLTQLFG
16
SLTMH ---------- AREDVVSACKENWEIFLDLP --- DVARTSLRPTVKNSQFMTPLFEFSGACCGTPYLKLLTQMWG
17
ALQMV ---------- SLDSQRAM-APVWDVALGLA --- PKDNPFRKTTVKGSQFETPLLEFSGACAGCGETPYARLITQLFG
18
TTNIDTESCNLRDLQLRFPYMEFPGSCPGCGesESTMKLATLYG
-LAMT ---------- PIDTVLDKQQKLFDWAYDSL
19
PIYTGRCGGAGRIKA
KA ---------------------------------- PVEEFYQSGHRT~CQLALPMRLMAKAAG
------21
MLiM---------------------------------- AQTEYFDAGHRTVCESAVLRWVAKAAG
-------22
23---------------------KTIKQA ----------------------------------PLEEFYASGHRTCGCEALLMRYLAKI SG
24---------------------KTIKDA ----------------------------------PLEEFYTSGHRTCGCESALLMRFLAKSAG
25---------------------KTIKDT ----------------------------------PLEEFYTSGHR~TQGCESALVMRFLAKAAG
26---------------------KSIAQA ----------------------------------PAEEYYLPGHRTCAUC6PALAYRLIAKAAG
---- KThftDM----------------------------------PQEEYYVPGHRTAiCPAVYRLVAKAAG
-27
U
29 (Mt QOR) -------------ASIKKA -------------------------------
---:-KT-----
PDEEYWVPGHRVCAC
TS:A------------------ PEEEYYLPGHRTCA
----------------
GALCYRLVAKAAG
GQFKELMEEHPMCSGW
YFIRLIFASLP
37
38
---- RK--------VD ----------------------------- 6QFKELVEEHPMCtAGCYMAYFIRVFYTALP
KKVSEM---------------------------------- GTFKEIIEEHPMCAGCAMTLFIRLTMIGLP
---- K ------ RGWKDV ---------------------------------- GSSTELIAEHSLCAGPER$IAFRHIMASIP
----- RGWOL ---------------------------------- PYTKELIEEHSLCAGCPESMALRYILASLP
----- ------ KK L---------------------------------- PFEEHFYAGHTACQGCGASLGLRYVLKAYG
- --- -------------------------------------------- APTATHYCAGCGHGILHKLIGEAID
--- AE ------------------------------------------------ ROYPFHYCPG0H6IIHRLVAEIID
39
--------
32
33
34
35
36
- ---
40
----------------------------
----------------------------------
DQPTWCNGCGDFGTMNGMMKALA
DLPTIFCPGCGIGAVLQYTLRAID
--- ST------------------------------------------- DiRMPHI&WCPC GTTVNCFSRALI
----------------------------------------------- TRIPHIWCPGCGIGTVFSSCLSAIK
DLPHIFCACGNG#IVLNTFFKGME
---------------------
41
42
43
46---------------------------------------------------------------KPNWCW6CGFSVQIKAAATI
--
47
48
50
------------
----------------------------------
SDQVRWCPGCGDYSVLGATRALA
------------------------------------------------------- VPNWCPGCGDFGVLKAKAMA
PRPSPAHYFAIKA
K---------------------------------V
---E
----INFA~~~~~1
LPRRPPVLCAGCGHRSTYYAVKLAA-A
--------------------- P1---51
---- AVPVD--TGEDFDYNV ---------------------------------- CRIDLPMRPPVMCPGCSHRATFYAMKKVY52
---- EKG ----------------------------------------------IEAPELPERPPALCPGCPIIRAMYVSVRRAAS
53
54---------------------------------------------------------------*TPQWNDWCPGCGWFGILNAEQQAIV
cons--*
130
Sequences42
PDTVLATPPGCVPGMGSVNTTGTKV---PVFHPLLTNTAAMLAGIKRQYKRVI ----------------------- RNSVLATPPGCIPGMGSV KTGTKV---PVFHPLLTNTASMLSGIRRVYDRIG -------------2
------ SNTILAIPPGCTAGVGAV4 GVTATKV---PSFHPLLTNTSSMLAGIKRYYNRI --------------3
---- ---YEPVVGLATGCLEVSTSI--YPYTAWSV---PYIHNAFENVAATMSGVETAYKALKNKG----------4
------ GPIVLGNSTGCLEVCSAV--YPHTSWDV---PWIHIGFENGSTAISGVEAMYKALVNKG ----------5
------ ENTIIVNATGCMEIIASQ--YPVTSWRL---PWIHTLFENTAAVASGVESAIKVLKRKK----------6
7
------ PNTIVINPTGCLEVMSTP--FPYSSWQV---PWIHSLFENAGAVASGVEAALKALG ----------------- KNTVAVSSTGCLEVITTP--YPETAWEI---PWIHVAFENAAAVASGVERALRARGU------------8
EMPNAFAIAQATCMEWSAV- -FPYTAWKV---PWVHVAFENAAAAASGVEAAWKKLG -------------9
10-----------------DQLYLANATGCSLVWGA- -TFPFNPFTTNERGHGPAWANSLFEDNAEFGGMFKAVEARRNITKKLVVELLIM
11-----------------ERMFIANATGCSSIWGA- -SAPS#PYKTNRLGQGPAWGNSLFEDAAEVGFGMNMSMFARRTHLAQLAAKALESD
12-----------------DRMIIAMATGCSSIWGG- -SAPACPVTV l~iPAWASSLFEDNAEFGYGMALAVAKRQDEL
------------- ERTIIANATCSSIWGA--TWGTNPYTV "PAWGNSLFEDNAEYGFGMFKANEQRRLYLEQICKE
13
14-----------------ERMVIANATGCSSIWGA--SVPSVPYTKNQKGYGPAWGNSLFEDNAEVGLGMVVGYRQRRDRFI LVSNEILKD
CILQAVE
15-----------------ERTVIANATGCSSIWGG--TAGLAPYTTNAK43QjPAWGNSLFEDNAEFGFGIAVANAQKRSRV
V
16---------------- DRLIIAINATGCSSIWGG- - SAPStPTTNADGWANSL FEDNAQFGLIAMGTMQRRK
RLGAA
17-----------------DRLMIANATGCSSIYGG--NLPTSPYAK A4GRGPAWSNSLFEDNAEFGLGFRLALDQHRSELT
SAPSMPYTTVRGHGPAWANSLFEDNAEFGLGMMLGGQAIRQQIAEE
18-----------------DRMLIAr4ATGCSSIWGA19-----------------DSMVASIGVGCSLVWMH- -FGYMRPFNLDSDSRGIAACSSLFEDNSVFGWGVALDRDAKRA4LREYLQANVEKI
------ PNTIFIGPTGCMYVANAti-EFLTSPYSV---PWHHTQLGGGGAAAIGIASALRTLMLKG-----20
------ PRTIVLGTT6CYVANTT--YMTTPWVV---PWMHTQLGAAGSAAVGTSAGLKALMRKG----------21
------ PNTIVIGATGCt4YVANTT--YYTTAWAL---PWIHTQLSGTGSAVVGTAAALKALIEKG ----------22
------ ERTIVVGATGCMVVANTT- -YVTTSWVV---PWVHTQLGGSGGAALGTAAALRALMRKG ----------23
------ PRTIVIGATGCI4VVANTT--VVTTSWVV---PWVHTQLGGSGAAALGTAAALKALMRKG ----------24
------ QRTIVIGATGCMYVANTT--VVSTSWIV---PWVHTQLGGSGAAALGTAAALRALMRKG ----------25
------ KNTIFIGPT6G11YVANTS--YACGPWAV---PWTHAQITNGGAVASGIEAAFKMMIKKG ----------26
------ PNTIFVGPTGCMYVANTS--YGCGPWAV---PWIHAQITNGGGVASGIEAAYKAMMRKG ----------27
------ KNTIFIGPTGCMYVANTS--YGCGPWAV ---- PWIHAQITNGGGVASGISAAFKAMIRKH ----------28
29 (Mt QOR)------------PNTIFIGPTGCMYVANTS--YGCGPWRV---- PWIHAQITNGGAVASGIEAAYKAMIRKK ---------------- PNTIFV6PTGCt4YVANTS--VGCGPWRV ---- PWIHAQITNGGAVASGIEAAYKALIRKX ----------30
----- QPEETVTLGTAGCGRLAIS---QAAV---PFIYGNYGDQNAMASGLTRAFRLtF -------------31
----- NPEDTIVIGTAGCARLALS---QAAV---PFIYGNYGDTNAVASGLKRALSIRF -------------32
----- NPEHTILVGTAGCGRLALS---QTSI---PFIYGNYGDTNAVASGLKRGLEMRF -------------33
----- APEDTVMVGSTGCTSLVFP---MVAV---HNIHSLFGNQNAVATGLKRALNVRF -------------34
IAL---HTVHSLFGNQNAVASGIKRVLEWRF ------------------ NPEDTIIVNSTGCTSLVFP --35
------ GKAIFTIPACCSTIIAGP--WPYTALNA---PLFHTAFETTGAVISGIEAALKAKGYN----------36
---- -ELGI-QERSVMISPVGCAVFAYY---YFDC---GNVQVAHGRAPAVGTGISRAED----------------37
FNC---DMLEAAHGRAPAVATGVKRVHP -------------------- ELGU-HDRAIGVAPVGCSVLAYE --38
MRS---YALHGVHGRALPVGTGVKLANP -------------------- ETGt4SPDDTFVVAGIGCSGKIGT --39
---- DLGLNQDEIVWVSGIGCSSRVPG---YVNF---DGLHTTHGRALAFATGIKLANP ----------------40
VNL---DSFHTTHGRAIPFATGLKLANP -------------------- ESCMNVNDMAIVSGIGCTGRVAG --41
DSFHTTHGRAIPFATGIKMANP ----------------IK ------ ATGIPYNKFTMVSGIGCSARGAG --42
VKC---DSLHTTHGRPIAFATGLKLANP ---------------------- MAGVDFDSIAMVSGIGCSSRIPG
43
---- ALGWKt4DDVCLVSGIGCSGRMSS---YVNC---NTVHTTHGRAVAYATGIKMANP----------------44
---- ELGLRRENIVFISGIGCSSRFPY---YLET---YGFHSIHGRAPAIATGLALARE ----------------45
INA---YGVHGIHGRALPLAQGVKMANR -------------------- NIGLEPEEVAIITGIGCSGRLSG --46
---- ELSIPPENVALVSGIGCSSRLPA---YTNV---FGFHGVHGRALPIATGIKVSRP ----------------47
---- ELGKDPEEILLATGIGCSGKLNS---YFDS---YGFHTIHGRSLPVARAAKLANH ----------------48
---- ELGLKPENIVSVSGIGCSSRLPL---FVKN---YSVHSLHGRAIPVAVGIKLARP ----------------49
---PIRT---VDTTVAMGASIGIG GLSIAMNGS
GPKAIYPSDIGCYTLGVLU
50--------- 1VKPVYANDIGCYTLGFYU-----PFEM---ADFTWSMGSALGIGMGiISKfS -----------------51
ITTLCMGGSITVASGMYQAGE ------------------------ GKDAIFPSDIGCYTLGIQ ----52
----ADVLLSMGSSVGTACGFSAAT -----------------PYSA
---- ELGIEGEDLIFPTDIGCYTLGIEE--53
FFRTE ---- SGVHTLHGRAIAFATGIKLSNP -------------------- ELGVDTKNVVWVSGIGCSGKIPH --54
1
cons*
131
Sequences
1
2
3
4
5
6
7
8
9
10
11
12
13
14
16
17
18
19
20
21
22
23
24
25
26
27
28
29 (Mt QOR)
30
31
32
33
34
35
36
37
38
39
4A0-41
42
43
44
45
46
47
48
49
50
51
52
53
54
10913
---RDVQALAIA6DGGASDVGFQSLSGRAERG-EQMLFMVVDNEGYM4TGMQRSSCTPY-GAWTSTTPVGETS- RGKTQDAK
- -- RKDIVVMALAGDGGTADCGFHAVSGAAERG-EKVLYVCVODiEGYKWTGMQRSGTTPY-GSWTSTTPVGEHM -QGKTQDAK
----GRDITMMAFAGDGGTADVGFQSLSGAAERG-EQMIYICID4EGYNTGVRSSTTPY-6AWTTTTPVGAL --KGKTRDAK
TGQSSP-STTPGK--PGKVQLKK
NEKYFAGDGYILSLGLR-KLVYNG
RYQGQRPKFVAPGGOGASYDIGQFISGCMEtG-HDMTYICLf4ENYMITGGQRSGSTPL -GASTSTTPAGSVS--FGKKEKKK
RIDDKVIGGTDGQLGMEGHFYCDEYNGQSAP-GASTTTSPAGKVG--KGQFSWKK
- - -- DDVKVVSIeGGGSTMDIGLGALSGAFERG-DTYVCMtDNAYMNTGQtSSTP-DASTTTTPAGKVS--FGNPRPKK
- -- -GEVNVVAFAGDGTADIGLQSLSGAMERG-t*IIYICYDNAYMNtT6IQRSASTPY-GASTTTSPHGKES--FGEDRPKK
TGIQRSSTPY-GATTTSPPGKYS- IGED.KPKK
- - -- RKGKILAIGOETADIWQALSGMLERR-HNVVYLMYDNEA
AOILSKKQVWIVGGGAYDIYGLDHYiASG-ENVKIUZ'0TEVYSTG4 SKATSR-AVANFSAAGYT---KAKK
SDLVTKKSVWIFGGDGWAYDIGYGGLDHVLASG- EDNVFVMDTWVYSNGGSATPT-GAVAKFAAAGKR---TGKK
KDLKSWIGGADIYGDVAGAVVLLTVSTGSKTQT-GAVARFAAGGKF---TKKK
KATNL-GAVVWFASSGCK---RPKK
SDMFVKISHWIVGOWwAvotGYNGVDHVLASG-NwVNLVLDTE1mSNTr
RDIKSWVGDWYIYGDVLF$EVII(TVSNGOSSTPF-GAIAKFAQSGNL---RQKK
YSTGGQRKATPK-SAVTKFAAGGKE---RPKK
RDMLHKASIWIVG*GW0Z6YIGDHVLASG-RffVfNfLfTE
NNLVDEYNGQSSP-GASAKFSVAGKA---LPKK
MYVKVIGDWYIGGLHIS
R---TRKK
RDFRSWFGGADGGLHLS-DNIVDEYNGQSSP-AAFA
N---VKDK
NNLKSWVGGADDAFHVS-RRVVVMFNGQSAS-SMNY
TRYKK
Z6f4DILSGLSMGLSYDQRLLYVYDNGYTGVQSSTTP-GATTTFTPSGKV
KRKPEDINVIV
KKN
KMKDEPINVIAFC20P0AD46LSAISAALTDIEVNLLIFLYDNESYANThIQTSGQTPW-GAVTTFSPPGKKF
KKN
KLPNEKINVIGK#0OLGADALSEVSNALTHTVNFLLMYD1NESSANTD$QfTTMTPY-GALTTFSPSGKQ
WKKS
KIKEEPINVIVFCGDLGCAbftGLSVSNMTYD-YNLLIILYWIESSANTDQTSMTiY-GAQTSFSRPGTA
KKS
KIKQEPINVIAFCGDLGCADGLGVSNAMTYD-YN4LULYSSATDQTSITPY-GAQTTFSRPGKVR
WKKS
KIKQEPINVIAFCGDLCADMLSGSNA4TYD-YLLIVLYDNESANITZtTSMTPY-GAQTSFSRPGKQR
AKKFFPK
KIKDEFPNIIV AGDGAIDGLQAASAMYRG-?0VLFIMYDNESYANTGIQTSPSTPY-GATTMFTPAGPEIKYTGERPHVIVMADG DGLQLSGZ~YR-4DVLFICYtDRESYANTGIQTSPTTPWd-GAVTTFTPSGPAV- GKRLWPK
KTDAEYPNIIVA0DGGADIGIQALSAAPYR-KWVICDNESYANTI APTTPY-GAWTTFTPAGPVV- EKKLMPK
TGITPTTPY-GANflFTPPGEVV- GKKLFPK
KTOAEFPNIIV, GGAVILALALR-HVFCYNSA
KDEPIVAOGVILASMYGHVFYDEYNGQSPTY-GAMflFTPPGPVV- EGKKLFPK
$GQSSP-GAItMAPLGKK---GEKM
PMIGFSTHWIGEFTILNVG
- -PEKHKDVITIAGDO
- -PDKHKDWVVIAGGGLIDGFMTMrnvvFR-EKFTTIMVNV$N4QSMtP-GVQLX APL6KK---FDKI
EKM
- -PDQSKDVVVMAGDGLADGFSTVLSWFRK- EKFTTIPML3NEVYWNTGGQSGER-GAILK4APKGKK ---- DTDIVADAVDGDT$&FGEFTIFNLANGQSLQK-GFVAKMAPVGKK---FEKV
- -PDKVKDVVVLAGDGAI~t)IGLDCTLQSFFRQ-EKITTICFOWEVYANGOE6RTVK-GHVFKMAPKGKQ---WDKV
NGIQSTPY-GAWTTNTPGGKKH--Fk.ERPKK
-EDIMVIGWAGbGGTADIGLASFRG-H1AEVWYDe:Ab
--- TPVVLLYQGDGLASIGLNETIQAANRG-EKMAVFFVNNTYGTGG ATTLI-G'EVTVTCPGGRDP- - GYPL
PIR
--- HAVTQDDAICEVAAGEITFNAYMGQATTLV-EQVTNTSPYGRKV-S-DP:ETSTSPDGPK---QPPV
--- DLEVMVAGGGYSIGA6HFIHAVRRN-VDITYVfaDRr2YriLK QSP
APTTiK-GLRGTTAPYGSF---ENPF
DLKVIAFMDGAAA4GGNLIIRt- LDVTVLZNNFYGMT
--- KRQVVVYSGDGDLFAIGESNHLIHAARRN-IDLKVICVNNLZYAMTGGTGPATPG-DVXTSTAPVGTF---DPAF
E
LKVIVFSGDGDLFAIGGNHFIAMRN-MD-LTVICVNNLTYGMTGGAA-TPS-M'AKSTTTPLGNP---DAPF
----- SLNVVVFTGDGAAAGGHLIHGAtKN-IDTVICINNSIZ Of4T4GQISPTSPE-GSFGTTAPGAL ---- EDPF
--- SKHVIWSGD~DGFIGGHTMHACR#4-IDLNFILVNFIYL1TTKLmRN-GWSVTAQWGNI---DNQF
--- DLSVWVVTGDGALSIGGNHtLIHALRRN-It4VTILLFMRI4LTKG YSTSV-GKVTKSTPMGSL ---- DHPF
--- DLTVIASGGDGDGYAIGM6HTIMiALRRN-MNMTYVIMNYL1TKG TSSSAP-GFVTKSTPKGNI ---- EKNV
--- ELTVIA$GVDSIGG*FMACRN-VD4TYIVDEVGTKG STTAPSWEKSKLTPQGTG ---- INPF
--- DLEWVAAGGQGDYIGNPMHTAREN-HIQTYIVNNVFGLTG %PT? I-GHKSKTQPHGSA---KSPI
4.GTK VSPT fE-GLYGSLTPYGSI---DRPV
--- DLTVIVETGDGDLFSIGAGHNPIIAARRN-IDXTVICMN
6PTGQ---TPHGM -------- NKRI
--- KQIIVATIGDSTFYHTGLPALAMAIYNR-SNVLIYVL*I. TATGD
AGEP---RPVV
--- KEPVIAFIGDSTFYHAGIPGLINAVYNR-IPLVV 4NIATKPPSGF-GP --TGEN---TVEV
THPPGMGK-TA----- KKPICCSIGD$i'PFHTGMNGLLNAZYNK-ADITVTIVNT
--- SQRIVSFIGDSTFFHAGIPPLINAVHNR-QRFVt.VIL0WRTTAMTGG HGLPV ---------4GEE---APAI
--- DLVVIVNGGDGDLLGIGAGHFVAAGRRN-VDMWILHDN.GVYGLTKGQASfLtKR-GEKPKSLPRPNI ---- NDAV
cons
I*
Site 4
132
m
225P
Sequences
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29 (Mt QOR)
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
cons
NLPLIM--VNHR-CAYVATASTA-YMED-------LYDKLDKAI-3 NG-FAYLHV-YSPCTTA-WRFPSN-LNMEV----NLPLIM--TAHK-CRYVATASTG-YMED- -LYEKLDRAIA-FEG FSYLHI YSPCPTG-WRIPSS-KVIDA----KG-MAYLHV-FSPCPTG-WRFPPE-KLIEV----YLPIIM--MMHN-CEYVATASTS-FMDD-------YYEKLDKAYE NIVEIV--AAHE-NVYAATASLS-EPMD-------FFAKVEKALN---FDG-PSFLAV-FSPCVRF-WRVNDD-KTVEI----DIVNIM--ASHG-VPYVAQLSPN-KWKD-------MNKKIKTALD---TEG-PCFINA-LSPCTTE-WKFDSN-KTIEL----DMPAIA--VAHN-IPYVATCCPS-YPFD-------MIEKVGKALA---ADG-PAYIHC-LSVCPTG-WRCATE-DTVKI----NMPAIM--AAHG-SPYVATTSIG-FPRD-------MIRKVKKATE--- IVG-PTYIHA-QAPCPTG-WGFDTS-KTLEI----NMPLIM--AAHG-VPYVATASIS-YPED-------FMEKVRKARD---IEG-PAYIHL-HQPCTTG-WGFDPS-KTVEL----WVALIA--AAHQ-VPYVATASIG-NPFD-------FVRKMKKAAK---VDG-PAFVQV-HCTCPTG-WKSPLE-KGVEI----DLGAIA--MTYK-NIYVASTCLLIDPNQ-------ALKALQEAKE-- -YNG-PAIIIN-YSPCINH KKGLG-STPKH-----
DLARMV--MTYG-YVYVATVSMGYSKQQ-------FLKVLKEAES---FPG-PSLVIA-YATCINQELRKGMG-KSQDV----DLGLMA--MSYG-YVYVASVAMGASHSQ-------LMKALIEAEK---YDG-PSLIIA-YAPCINH--GINMT-YSQRE----DLGAIA--MAYG-DVYVASIALGANPAQ-------AFKAFKEAES--- YNG-VSLIIA-YCPCKEQ--GVPLQ-KSIEE----DIGSIA--MEYG-SVYVASVALGANYSQ-------TIKSLLEAEK---YPG-TSLIVA-YSTCIEH-GYTKYN-LQQES----NLTEMA--MSYG-NVYVATVSHG-NMAQ-------CVKAFVEAES---YDG-PSLIVG-YAPCIEHILRAGMA-RMVQE----DLGAIA--MSYG-DVYVASTSLHANYGQ-------VTKAMAEAEK---YGG-VSLVLA-YAPCIMHEISSGMC-SAIDE-----
DLGQIA--MANG-HVYVASIAFGASDNQ-------TLRALSEAVS---YEG-PSLIIA-YSHCIAH--GYDLT-CGLSQ----DLGMMA--MSYG-NVYVAQIAMGADKDQ-------TLRAIAEAEA---WPG-PSLVIA-YAACINHELKAGMR-CSQRE-----
KMGVML--MSTYRDVYVAQISLGYNRNQ-------ALQVFREAEH---FDG-PAVVLA-YCPCISHEQGGLT-HQERQ----NVALIA--ASHPNVKYVATATLW-PPLD-------LMNKVVRALN---SGG-PSVIHA-LFPCPKG-WFTSPA-DTVML----VPGMLA--AGHPDTRYVASGTPA-IPVD-------MMDKVRAALR-- AGG-PTYIHV-LDPCPKG-WHYDPE-HSNEL----MAAMMA--VGHPEVRYVATATAA-DPID-------LYNKVKKALE-- IGG-PTFIHT-LDPCPKG-WGYDPK-YSHEI----VVPMII--AGHRNVRYAATTSPA-YPLD-------TLNKIKKALS--- AGG-PTFIHS-LDPCPKG-WDYDPM-YSHEL----VVPMII--AGHRTIKYAATMNPA-YPLD-------GLNKIRKALE-- IGG-PTFIHS-LDPCPKG-WDYDPQ-FSHEL----VVPMII--AGHRNIRYAATMTPA-YPLD-------SINKIKKALA---IGG-PTFIHS-LDPCPKG-WDYDPR-FSHEL----GHPEVKYGATASVA-YPVD-------LMNKVRKGLA-- YEG-PAFLHV-QCPCPKG-WTFPAD-KTVEI----DPMAIF
GHPELKYLATASPA-FPVD-------LFNKVRKGLN---AEG-PAYLHI-QAPCPKG-WQFPSS-KTIEM----DNAQMIGHPELKYLATASLG-YPLD-------LMNKVRKACN---AEG-PAFLYI-HAPCPKG-WSFPAE-KTVEV----DITKVMGHPELKYVATASIG-WPVD-------LMNKVRKGLN--- QEG-PAYIHIIHAPCPKG-WQFPAD-KTIEM----DNPKVIDNPKVV- GHPELKYVATASIG-WPVD-------LMNKVRKGLN---QKG-PAYLHI-HAPCPKG-WQFPAN-KTIEM----PATALA--QAAG-CVYTVRLSPT-NIKK-------AAKVIRRAIF REVG-PTFVHA-YTSCNIE-YSIPTE-DVFAD----NAVELA--KTAG-CVYVARISPT-NPKR-------IAKTIRRAIL RHFG-PTFIHA-YTSCNIE-YSIPTR-EVLAD----EIGPTFIQAYTSCNIE-YAIPTP-KVIDD----DMLGLA--KTAN-VPYIVRMTVT-NPTR-------VASVVRKAVL
EIGPTYLI-YTPCILE-IGKQSM-EGLQE----RLPEIA--RESG-CAYVAVLTTS-KPSL-------VEKVVRQAVL
PMWQIA--IDSG-CHYVARLTVS-SPKR-------VESVIKKAIYREVGPTYVHLYTPCILE-IGLNSD-DGLDE----KVIDIV--IAHK-PAYAATASIA-YPED-------FMRKLKKAQT---IKG-PSFIQL-FAPCPTG-WRSPTD-KTIEL-----
HMCELL--DNLQAPVFIERVSLA-DPKS
LEIQRD---
GKGYAFVEVLSPCPTN-LRQDAE-GAERF-----
rfKAFEILN--- GEG FALVEV-LSTCPTN-WGMTPL-EAVDW ---VSEMLV- -TLDG-AKYIARVAVN- NPANN
NPLALA--MAAG-GSFIAQSFSS-DALR-------HQEIVQEAIE---HDG-FSLVNT-FSPCVTF-NDVDTY-DYFR--DIAELA--VAAG-ANYVARWSVF-NYLQ-------GINSIKKALQ---KRG-FTLVEF-LSQCPIS-FGRRNREKTG
--NLPYLT--EAAG-AVYVARWTTF-HVRQ-------ITKTMQEMFS---KKG-FCFLEI-VSPCPTL-YQRRNK
--NLPLLA--YAAG-ASYVARWTIL-QTRD-------LTTAINEAIS---RKG-FSFIEV-LSPCPIN-YGRRNKEK
DLSELV--RAAG-ASYVARWTAA-HPLQ------- LANSIKKGLK--- NRGFSFIEAVSQCPTY-FGRKNR
DPCALT--TAAG-ASFVARESVL-DPQK-------LEKVLKEGFS-- HKG-FSFFDDIHSNCHIN-LGRKNK
--NPVSLA--LGAE-ATFVGRALDS-DRNG-------LTEVLRAAAQ---HRG-AALVEI-LQDCPIF-NDGSFD
APLELA--LSSG-ATFVAQGFSS-DIKN-------LTKLLEDAIN--- HDG-FSFVNV-FSPCVTY-NKVNTY-DWFK
HPLVIA--LAAG-ANFIARGFSG-DPNG-------AAQLITEAIR--- HPG-FSFVQL-LSPCVTF-RPEQRD-WKDA
RPLSLS--MTSG-ASYVARTAAV-NPNQ-------AKDILVEAIQ-- HDG-FAHVDF-LTQCPTW-NKDAKQ-YVPY
--NPIATM--LSYG-ATFVAQTYAG-NLKH-------MTEVIKQAIQ--- HKG-FSFVNV-ISPCPTF-NKVDF
PIEDVA--KAMG-ADFVAVVDPY-DIKA-------TYETIKKALE-- VEG-VSVVVS-RQVCALY-KIGQ
AVVVSRRPCALM- ELRRKR
KIEDIA - -KAVG-VEFVEVVDAY-DVPA ------- VRDAVERAIRP
SLEALC--RGLG-AEFVEVVDAY-DLEQ-------TEEVFKRAKS---YKG-TSVVIT-KQLCVID-AKRSGI RR
SIEDIT--RACG-VEFVETVNPM-NIRR-------SSETIRRALQ---HES-VAVVIS-RYPCMLS-EGAVRG RPV -NPIALA--ISSG-YTFVARGYAY-DVKH-------LKELIKSAIK---HKG-LALIDV-LQPCPTY-NDINTK-EWYDK ---
El
m .3
.
I
*
I
I
m
133
134
Marcus Ian Gibson
77 Massachusetts Avenue, Bldg. 68-688
Cambridge, MA 02139
migibson@mit.edu 1510-292-7454
CURRICULUM VITAE
EDUCATION
Ph.D. Inorganic Chemistry - June 2015
Thesis: Structures of Oxalate Oxidoreductase: C2 Activation by a Microbial TPP-Dependent Ferredoxin
Oxidoreductase
Massachusetts Institute of Technology (Cambridge, Massachusetts)
Research Advisor: Professor Catherine L. Drennan
B.S. Chemistry - May 2008
University of California, Berkeley (Berkeley, California)
Research Advisor: Professor Jeffrey R. Long
AWARDS
2013
Martin Family Fellow for Sustainability
2012
National Science Foundation Graduate Research Fellow
2009 - 2012
Fall 2008
Howard Hughes Medical Institute Teaching Assistant Fellow
RESEARCH
-
EXPERIENCE
Graduate Student Researcher
Nov. 2008 - Present
Advisor: Professor Catherine L. Drennan (MIT)
Project: Biophysical characterizationof metalloenzymes in the Wood-Ljungdahl Pathway of acetogenesis.
Description: Enzymes involved in one-carbon chemistry are of interest for a number of applications from
greenhouse gas reduction to biofuels production. Using X-ray crystallography, I determined structures of
an oxalate oxidoreductase (OOR) from Moorella thermoacetica,which is only the second enzyme from the
2-oxoacid:ferredoxin oxidoreductase enzyme superfamily to be structurally characterized. I also performed
solution studies of the formation of a complex of enzymes from M thermoacetica that is responsible for
transferring a methyl group in acetyl-CoA synthesis.
Collaborators: Stephen W. Ragsdale, Elizabeth Pierce, and Mehmet Can (University of Michigan)
Project: Structuralstudies of multi-heme cytochromes involved in dissimilatory metal reduction in Shewanella
oneidensis MR-i.
Description: Multiheme cytochromes in S. oneidensis allow the organism to grow on metal surfaces, which could
have important applications in the development of biofuels. I expressed and purified a decaheme cytochrome
involved in electron transfer that I then used for further studies intended to determine the structure of the
protein and the arrangement of the heme cofactors.
Collaborators: Sean Elliott and Mackenzie Firer-Sherwood (Boston University)
135
Marcus 1. Gibson
Graduate Student Researcher
Aug. 2008
Advisor: Professor Jonas C. Peters (MIT)
Project:Synthesis and characterizationtransition-metal-basedhydrogen reduction catalysts.
Description: Hydrogen gas is a potential alternative fuel to fossil fuels, but current methods to produce it require
significant over-potentials, making it too expensive. As a step toward making a more efficient hydrogenproduction catalyst, I developed a synthetic strategy and characterized glyoxime-based ligands to coordinate
metal ions such as iron, cobalt, and nickel, which would be useful for understanding how ligand substituents
affect a catalyst's ability to reduce protons to hydrogen gas.
Undergraduate Research Assistant
May 2006 - May 2008
Advisor: Professor Jeffrey R. Long (UC Berkeley)
Project:Synthesis and characterizationof novel metal organicframeworks (MOFs)for the purpose of hydrogen
storage.
Description: Hydrogen gas is a potential alternative fuel to fossil fuels, but currently there is no efficient method
for storing hydrogen gas that would enable it to replace fossil fuels in small vehicles. I was the first person
in the lab to work on synthesizing MOFs containing magnesium and beryllium, which have a potential to
store large quantities of gas like a sponge. Over the course of my time, I contributed significantly to the
lab's knowledge of how solvent impacted framework structure, as well as the important role of water in
framework synthesis.
PUBLICATIONS
"Structure of an oxalate oxidoreductase provides insight into microbial 2-oxoacid metabolism" Gibson, M. I.;
Brignole. E. J.; Pierce, E.; Can, M.; Ragsdale, S. W.; Drennan, C. L. (submitted).
"Intermediate structures of oxalate oxidation suggest a bait-and-switch mechanism in thiamine-dependent oxalate
oxidoreductase" Gibson, M. I.; Johnson, A. J.; Brignole, E. J.; Pierce, E.; Can, M.; Ragsdale, S. W.; Drennan, C. L.
(in preparation).
"p[2-Iodido-bis-dimethyl [m ethyl -bis(quinolin - 8-y I)si lanyl--K3N, Si,N'] platinum(IV)tetrakis(pentafiuorophenyl)borate
dichloromethane 0.66-solvate" Carr, C. A. M.; Gribble, C. W.; Gibson, M. I. Acta Cryst. (2008), E64, m472.
ORAL PRESENTATIONS
Chemistry Student Seminar, Department of Chemistry, MIT
Jan. 2015
Chemistry Student Seminar, Department of Chemistry, MIT
May 2014
Inorganic Chemistry Departmental Seminar, Department of Chemistry, MIT
Jan. 2014
POSTER PRESENTATIONS
Chemistry Student Seminar, Department of Chemistry, MIT
Jan. 2015
Chemistry Student Seminar, Department of Chemistry, MIT
May 2014
Inorganic Chemistry Departmental Seminar, Department of Chemistry, MIT
136
Jan. 2014
Marcus I. Gibson
TEACHING
EXPERIENCE
Massachusetts Institute of Technology
Aug. 2012
June 2015
-
Graduate Resident Tutor (McCormick Hall)
Description: Resident mentor, tutor, first-responder, and community builder for
36 undergraduate students in MIT's all-women dormitory.
Supervisors: Professor Charles Stewart III and Kathryn Hess
Teaching Certificate Program
This course is provided through the MIT Teaching and Learning Laboratory. A
letter of support and a copy of my teaching philosophy are available upon request.
Spring 2014
Undergraduate Research Mentoring
Feb. 2012
-
Aileen Johnson (MIT undergraduate; currently an M.D. student at Emory
University School of Medicine)
May 2014
Project: Crystallization of a thiamine-dependent oxidoreductase from Moorella
thermoacetica.
Project: Cloning and expression of a multi-heme cytochrome from Shewanella
oneidensis.
Evan Waldron (Vassar College undergraduate; currently an M.D./Ph.D. candidate
at Rutgers New Jersey School of Medicine)
Summer 2010,
Summer 2011
Project: crystallization of multi-heme cytochromes from Shewanella oneidensis.
Sep. 2009
-
Andrew Van Benschoten (MIT undergraduate; currently a Ph.D. candidate at the
University of California, San Francisco)
May 2010
Project: Structure and function of nickel regulatory proteins.
Teaching Assistant andAcademic Tutor
5.78: Biophysical Chemistry Techniques
Fall 2012
Laboratory Teaching Assistant
Instructor: Prof. Catherine L. Drennan
5.03: Principles of Inorganic Chemistry I
Spring 2010
Department Tutor
Supervisor: Melinda Cerny
5.310: Laboratory Chemistry
Spring 2009
Laboratory Teaching Assistant, Guest Lecturer
Instructor: Dr. John Dolhun, Instructor
5.111: Principles of Chemical Science
Fall 2008
HHMI Teaching Assistant Fellow
Instructors: Prof. Catherine L. Drennan, Dr. Elizabeth Taylor
137
Marcus I. Gibson
REFERENCES
Catherine L. Drennan
(Research Advisor)
cdrennan@mit.edu
Stephen W. Ragsdale
HHMI Professor and Investigator
Departments of Chemistry and Biology
Massachusetts Institute of Technology
77 Massachusetts Avenue
David Ballou Collegiate Professor of Biological Chemistry
Department of Biological Chemistry
University of Michigan Medical School
1150 W. Medical Center Dr.
Room 68-680
Cambridge, MA 02139
(617) 253-5622
5220D MSRB III
Ann Arbor, MI 48109
(734) 615-4621
Stephen J. Lippard
(Thesis Committee Chair)
Sean J. Elliott
lippard@mit.edu
elliott@bu.edu
Arthur Amos Noyes Professor of Chemistry
Department of Chemistry
Massachusetts Institute of Technology
77 Massachusetts Avenue
Associate Professor of Chemistry
Department of Chemistry
Boston University
590 Commonwealth Avenue
Boston, MA 02215
Room 18-498
Cambridge, MA 02139
(617) 253-1892
Charles Stewart III
(Housemaster)
cstewart@mit.edu
(Collaborator)
sragsdal@umich.edu
(Collaborator)
(617) 358-2816
Kathryn M. Hess
(Housemaster)
kmhess@mit.edu
Kenan Sahin Distinguished Professor of Political Science Environmental Scientist at the
Department of Political Science
U. S. Environmental Protection Agency
and
and
Housemaster of McCormick Hiall
Housemaster of McCormick Hall
Massachusetts Institute of Technology
14 Hurlbut St.
77 Massachusetts Avenue
Cambridge, MA 02138
Room E53-447
Cambridge, MA 02139
(617) 253-3127
138