The Structure and Function of
-Glucosidase from Human Gut Bacterium
Ruminococcus Obeum
Kemin Tan and Andrzej Joachimiak
2009 NIGMS Workshop: Enabling Technologies for Structural Biology
March 4-6, 2009
Gut Microbiota
Division
Actinobacteria
Bacterioidetes
Firmicutes
Proteobacteria
…
etc.
{
Genus
Clostridium
Eubacterium
Ruminococcus
…
etc.
Species
Strain
{
Gnavus
obeum
…
etc.
ATCC 29174
Known Functions
 Maturation
 Development of innate immunity
 Production of essential vitamins
etc.
Nondigestible food components serve as sources of energy and
carbon for the human gut bacteria.
The Journal of Nutrition. 2007
Over-Represented Genes
Statistics for Some Genomes by COG Catagories
Genome Name
Bordetella parapertussis 12822
Corynebacterium diphtheriae
Cytophaga hutchinsonii
ATCC 33406
Enterococcus faecalis V583
Escherichia coli K12
Geobacter sulfurreducens PCA
Haloarcula marismortui
ATCC 43049
Listeria innocua
Methanocaldococcus jannaschil
DSN 2661
Porphyromonas gingivalis W83
Pseudomonas syringae pv.tomato
str.DC3000
Ruminococcus obeum
ATCC 29174
Silicibacter pomeroyi DSS
Sulfolobus solfataricus P2
COG genes
3654
1576
2226
MetabAA
490
174
163
Percent
13.41%
11.04%
7.32%
MetabCarb
197
107
126
Percent
5.39%
6.79%
5.66%
MetabLipid
239
57
97
Percent
6.54%
3.62%
4.36%
2210
3566
2527
2642
180
367
190
268
8.14%
10.29%
7.52%
10.14%
262
377
99
140
11.86%
10.57%
3.92%
5.30%
61
103
64
95
2.76%
2.89%
2.53%
3.60%
2391
212
8.87%
278
11.63%
59
2.47%
1427
1233
4177
106
78
458
7.43%
6.38%
10.96%
51
59
261
3.57%
4.82%
6.25%
14
40
180
0.98%
3.27%
4.31%
2393
224
9.36%
245
10.24%
61
2.55%
3399
2105
5561
202
6.36%
9.60%
204
128
6.00%
6.08%
194
86
5.71%
4.09%
Thermoplasma volcanium GSS1
Vibrio parahaemolyticus
RIMD 2210633
1214
3259
117
346
9.64%
9.80%
89
207
7.33%
5.87%
50
123
4.12%
3.49%
Glycosyl Hydrolases
In Ruminococcus obeum ATCC 29174,
245 genes in carbohydrate transport and metabolism,
22 genes as glycosyl hydrolases (GH).
GH1, 1
GH2, 2
GH3, 3
GH18, 1
GH20, 1
GH31, 1 (-glucosidase)
GH32, 4
GH42, 3
GH43, 4
GH77, 4
Crystal Structure Determination
X-ray Diffraction Data Collection and Processing:
Data collection
SBCcollect
APS, Structural Biology Center, 19ID beamline.
HKL3000 program suite
data integration and scaling.
Structure Determinaion:
HKL3000 program suite
50 out of 54 Se sites located and used in phasing.
46 sites Se sites matched NCS and used for averaging and
phase improvement
Phasing
RCullis (anomalous) (%)
Figure of merit (%)
85
20.3
Refinement
Resolution
Reflections (work /test)
Rcry stal/Rfree (%)
Rms deviation fro m ideal geometry
Bond length ()/angle (
35.2 -1.95
99024/5209
17.18/22.19
0.015/1.486
3 cycles of Arp/warp model building: 1244 out 1332 residues
No.of atoms (Protein/HETATM)
Mean B-value (2)
(ma inchain/sidechain)
11758/856
12.77/15.15
sequence docked: 1211 residues.
Ramachandran plot statistic (%)
Residues in mo st favored regions,
in additional allo wed regions,
in generously allowed regions,
in disallowe d region
Model Building:
HKL3000 program suite
built (93.4%).
88.6
11.0
0.3
0.1
Dimer Structure in Crystal and Solution
Calculated monomer molecular weight: 77.4kD,
including vector derived residues.
Homologous Structures
Human intestinal maltase-glucoamylase
PDB: 2QLY
Overall sequence identity: 28%
Sulfolobus solfataricus -Glucosidase
PDB:2G3M
Overall sequence identity: 26%
8
Human Intestinal MGAM and SI
-Amylase
(endohydrolase)
amylose
Maltase-Glucoamylase(MGAM)
(1-4) high activity
amylopectin
Sucrase-Isomaltase(SI)
(1-6)
(1-4)
(1-4)
(exohydrolases)
Glucose
Catalytic Site
Catalytic domain
R.obeum -glucosidase: 366 a.a.
Human NtMGAM: 362 a.a.
Structural alignment:
310 a.a. aligned
RMSD: 1.68Å
Sequence identity: 29.6%
Catalytic nucleophile: the residue D307 in magenta.
Acid/base catalyst (possible): the residue D420 in green.
Substrate Hydrolyzed (mM)
Substrate Specificity
Maltose Sucrose Lactose
At least a maltase
Access to Catalytic Site
Glucoamylase ?
N- and C-terminal Domains
A Common Enzyme in Gut Microbiota
Coprococcus eutactus ATCC27759
Clostridium sp. L2-50
Clostridium phytofermentans ISDG
Faecalibacterium prausnitzii M21/256%
Clostridium botulinum c str. Eklund
Clostridium perfringens CPE str. F4969
………..
Petrotogo mobilis SJ95
Iden.
67%
69%
59%
72%
53%
54%
Posi.
80%
80%
75%
1%
71%
70%
Gap
0%
0%
0%
46%
65%
2%
2%
1%
14
Glycosyl Hydrolases
Maltase-Glucoamylase(MGAM)
amylose
(1-4) high activity
Sucrase-Isomaltase(SI)
(1-6)
(1-4)
(1-4)
(exohydrolases)
amylopectin
glucose
Conclusions
1. PDB: 3FFJ
2. Member of gut microbiota can also utilize digestible carbohydrates.
3. Potential competition between gut micobiota and human host in
utilization of carbohydrate resources.
4. Regulation ?
……
16
Acknowledgements
 ANL/MCSG
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A. Jochimiak
H. An,
G. Babnigg,
L. Bigelow,
A. Binkowski,
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C. Giometti,
W. Eschenfeldt,
Y. Fan,
C. Hatzos,
R. Hendricks
G. Joachimiak,
H. Li,
L. Keigher,
Y-c. Kim,
N. Maltseva,
E. Marland,
S. Moy,
R. Mulligan,
B. Nocek,
M. Schiffer,
A. Sather
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R. Wilton
Univ. of Virginia
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H. Zheng
Univ. College
London @ EBI,
J. Thornton,
C. Orengo,
M. Bashton,
R. Laskowski,
D. Lee,
R. Marsden,
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A. Todd,
J. Watson
Northwestern Univ.
W. Anderson,
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G. Minasov,
L. Shuvalova,
X. Yang,
Y. Tang
G. Montelione,
Ruthgers Univ. NESGC
T. Terwilliger,
Los Alamos, ITCSG
Z. Derewenda, Univ.
of Virginia, ITCSG
Z. Dauter, NCI
J. Liang, Univ.
of Illinois
D. Sherman, U. Michigan
Washington
Univ.
D. Fremont,
T. Brett,
C. Nelson,
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Univ. of Toronto
A. Edwards,
C. Arrowsmith,
A. Savchenko,
E. Evdokimova,
J. Guthrie,
A. Khachatryan,
M. Kudrytska,
T. Skarina,
X. (Linda) Xu
Univ. of Chicago
O. Schneewind,
D. Missiakas,
Univ. of Texas
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SWMC
S. Koide, ITCSG
Z. Otwinowski, W-j. Tang,
D. Borek,
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A. Q. Mei,
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M. Rowicka
T. Kossiakoff, ITCSG
V. Tereshko,
Funding: NIH and DOE
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7
Thank you