Acetyl-coenzyme A Synthase: A Novel
Organometallic Catalyst and Drug Target
Presented by: Seth Cory and Trang Nguyen
TAMU Fall 2014
A Cluster: active site of ACS reaction
Proximal Ni: (trigonal planar)
+ where substrate binds
+ very labile
 can be removed by
phenanthroline
Proximal Ni: (trigonal pyramid)
can be replaced by Zn and Cu
 deactivates ACS
Ni
2
Lindahl, P. Coordination & Bioinorganic Chemistry Lectures, Nickel Enzyme, Texas A&M
University, College Station. TX, USA, 2014
ACS/CODH Overview of Reaction
How CO is delivered from
C-cluster to A-cluster?
Active site of C-cluster
Proposed Mechanism of C-cluster
3
Macharak, P; Harroop, T. Coord. Chem. Rev. 2005, 249, 3007-3024.
Crabtree, R. The Organometallic Chemistry of the Transition Metals. 2005.
Structure of ACS/CODH
Darnault, C, et al. Nat. Struct. Biol. 2003, 10, 271-278.
BUT… the assembly chemistry of ACS is
still unknown!!!
4
ACS/CODH Overview of Reaction
????
Corrinoid Fe4S4 protein
diffusion
What is the interface domain between
CoFeSP and A cluster?
5
Closed conformation
Open conformation
Why do we study these interactions?
Understand the chemical mechanism of A-cluster
CH3Co3+-CoFeSP
2+/1+
S
[Fe4S4]
Ni0
S
Co1+-CoFeSP
2+/1+
N
S
[Fe4S4]
Ni2+
S
N
O
O
CoA
H 3C
H 3C
Ni2+
S
Ni2+
Step 1:
Oxidative elimination
with CH3- (Ni0 Ni2+)
S
S
CO
CoAS
2+/1+
[Fe4S4]
H 3C
-
S
Ni2+
Migratory Insertion
S
N
S
N
S
[Fe4S4]
H 3C
Ni2+
O
2+/1+
O
N
Dimagnetic
Mechanism
N
O
Step 2:
CO association (Ni2+)
CO
Ni2+
S
N
6
Ni2+
S
Adapted from: Lindahl, P. Met. Ions Life Sci. 2009, 6, 133-150.
N
O
Why do we study these interactions?
Understand the chemical mechanism of A-cluster
-
Ni2+ is activated by a 1e reduction by ferredoxin
Paramagnetic
Mechanism
CO
2+
S
CO
Ni1+
[Fe4S4]
S
O
S
CoAS
S
[Fe4S4]
Ni1+
N
CoA
N
S
Ni2+
H 3C
S
2+
N
Ni2+
S
Step 1:
CO association (Ni1+)
O
N
CH3Co3+-CoFeSP
Internal
e transfer
-
O
Co1+-CoFeSP
2+
[Fe4S4]
H 3C
S
Ni2+
2+
S
N
Ni2+
O
S
N
H 3C
Migr
O
S
[Fe4S4]
atory In
sertio
n
2+
S
[Fe4S4]
H 3C
Ni2+
CO
Ni3+
S
S
N
Ni2+
S
CO
Step 2:
Oxidative elimination
with CH3(Ni1+ Ni3+)
N
O
N
7
Ni2+
S
N
O
Adapted from: Ragsdale, S; et. al. Chem. Rev. 2014, 114, 4149-4174.
Why do we study these interactions?
Understand the chemical mechanism of A-cluster

8
Monsato Acetic Acid
Acetyl CoA Synthase
OXIDATIVE ADDITION with methyl group is a rate-limiting step
Temperature: 150-200 oC
Pressure: 30-60 bar
 Temperature: 60-65 oC
Pressure: 1 bar
How can we find the answer????
Lindahl, P. Coordination & Bioinorganic Chemistry Lectures, Nickel Enzyme,
Texas A&M University, College Station. TX, USA, 2014
9
Why do we study these interactions?
Understand the chemical mechanism of A-cluster

Develop novel catalysts in
industrial reactions
TAMU
CHEM 462

10
Proposals for studying Acetyl CoA Synthase
Aim 1: To study the methyl transfer from CoFeSP to
ACS active site
Aim 2: To probe coordination sphere of the A cluster
Aim 3: To study the dynamics ACS/CODH enzyme
11
How can we study these interactions?
Ando, N.,et al. J. Am. Chem. Soc.
2012, 134, 17945–17954.
Darnault, C, et al. Nat. Struct. Biol.
2003, 10, 271-278.
Biophysical Techniques to study the interactions
12
Ribonucleotide Reductases: A Model Study
Experimental
solution
scattering
Electron
Small Angle
X-ray
Microscopy
X-ray Scattering
crystallography
13
Ando, N; et al. Proc. Natl. Acad. Sci. USA. 2011, 108, 21046-21051.
Electron Microscopy
Small Angle Xray Scattering
Molecular Envelopes
14
Barondeau, D. Physcial Methods in Biological Chemistry, Structural Techniques, Texas
A&M University, College Station. TX, USA, 2013
X-ray Crystallography
15
Barondeau, D. Physcial Methods in Biological Chemistry, Structural Techniques, Texas
A&M University, College Station. TX, USA, 2013
How can we study these interactions?
Ando, N.,et al. J. Am. Chem. Soc.
2012, 134, 17945–17954.
Darnault, C, et al. Nat. Struct. Biol.
2003, 10, 271-278.
Biophysical Techniques to study the interactions
Electron Microscopy (EM)
Structural insight
Small Angle X-ray Scattering (SAXS)
at the interface
X-ray Crystallography (XTAL)
of CoFeSP & ACS
16
How can we study these interactions?
Ando, N.,et al. J. Am. Chem. Soc.
2012, 134, 17945–17954.
Darnault, C, et al. Nat. Struct. Biol.
2003, 10, 271-278.
Docking Model between CoFeSP & ACS/CODH
Site-directed Mutagenesis at the interface
17
Proposals for studying Acetyl CoA Synthase
Aim 1: To study the methyl transfer from CoFeSP to
ACS active site
Aim 2: To probe coordination sphere of the A cluster
Aim 3: To study the dynamics ACS/CODH enzyme
18
Site-directed Mutagenesis of A-cluster
19
Fontecilla-Camps, Handbook of Metalloproteins. 397-412
Site-directed Mutagenesis Procedure
Design primers with site-directed mutagenesis
Polymerase Chain Reaction (PCR)
DpnI Digestion & Transformation to E.coli cells
DNA sequencing to confirm the site-directed mutagenesis
Grow cells in large scale to obtain cell lysates
PURIFY the proteins (variants)
20
Site-directed Mutagenesis of A-cluster
1. Functional Assays:
 Acetyltransferase activity
 Acetyl CoA synthesis reaction rate
2. Kinetics with Crystallography
21
Structure of ACS/CODH
𝛂 subunit
𝐀𝐀𝐀
Closed conformation
Tunnel is open
 Binding of CO
𝛃 subunit
𝐂𝐂𝐂𝐂
CO
What are the dynamics
of the protein?
𝛂 subunit
𝐀𝐀𝐀
Open conformation
 Tunnel is obstructed
 NO binding of CO
23
Proposals for studying Acetyl CoA Synthase
Aim 1: To study the methyl transfer from CoFeSP to
ACS active site
Aim 2: To probe coordination sphere of the A cluster
Aim 3: To study the dynamics of ACS/CODH enzyme
24
Docking Model between ACS & CoFeSP
Predict the binding domain between CoFeSP & ACS/CODH
A
A
Strategy: Fluorescence Labeling at Specific Residues
a) Fluorescence Resonance Energy Transfer
25
Docking Model between CoFeSP & ACS/CODH
Predict the binding domain between CoFeSP & ACS/CODH
Strategy : Fluorescence Labeling at Specific Residues
a) Fluorescence Resonance Energy Transfer
b) Fluorescence Anisotropy
26
Docking Model between CoFeSP & ACS/CODH
Closed conformation
Open conformation
Experimental Techniques to study:
 Fluorescence Resonance Energy Transfer
 Fluorescence Anisotropy
27
Conclusion
Understand the methyl
transfer from CoFeSP to ACS
Understand
the chemistry
Understand the dynamics of
ACS
of ACS
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How serious is CDI?
 Signs & Symptoms:




Severe diarrhea
Colitis
Toxic megacolon
Intestinal perforations
 Current Treatments:
 Vancomycin: inhibitor of bacterial cell wall synthesis
 Metronidazole: inhibitor of bacterial DNA synthesis
Antibiotics
Resistance 
A drive to find
new target for the
treatment of CDI
McCollum DL, Rodríguez M. Clin. Gastroenterol. Hepatol , 2012, 10, 581-592.
29
Acetyl CoA Synthase: a new target for CDI treatment
3 potent inhibitors to inactivate ACS  potent to C.difficile:
1,10-phenanthroline
8-hydroxyquinoline
Inhibit methyl
transfer
Zhu, X. et al. Metallo., 2013, 5, 551-558.
2,2-dipyridyl
New antibiotics
for CDI treatment
30
Overview
 Understanding the chemistry of ACS is
important to the following fields:
Bioinorganic Chemistry
Biomimetic/Organometallic Catalysis
Medicinal Chemistry
 Several biophysical and biochemical studies need
to be done
TEM, SAXS, X-ray crystallography
Site-directed mutagenesis
Fluorescent studies
29
McCollum DL, Rodríguez M. Clin. Gastroenterol. Hepatol , 2012, 10, 581-592.