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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. Genus. Species. - PowerPoint PPT Presentation
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Kemin Tan and Andrzej Joachimiak
2009 NIGMS Workshop: Enabling Technologies for Structural BiologyMarch 4-6, 2009
The Structure and Function of -Glucosidase from Human Gut Bacterium
Ruminococcus Obeum
Gut Microbiota
Division
Actinobacteria
Bacterioidetes
Firmicutes
Proteobacteria…etc.
{Genus
ClostridiumEubacteriumRuminococcus…etc.
{Species
Gnavusobeum…
etc.
Strain
ATCC 29174
Known Functions
Maturation Development of innate immunity Production of essential vitaminsetc.
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 COG genes MetabAA Percent MetabCarb Percent MetabLipid PercentBordetella parapertussis 12822 3654 490 13.41% 197 5.39% 239 6.54%Corynebacterium diphtheriae 1576 174 11.04% 107 6.79% 57 3.62%Cytophaga hutchinsonii 2226 163 7.32% 126 5.66% 97 4.36%
ATCC 33406 Enterococcus faecalis V583 2210 180 8.14% 262 11.86% 61 2.76%Escherichia coli K12 3566 367 10.29% 377 10.57% 103 2.89%Geobacter sulfurreducens PCA 2527 190 7.52% 99 3.92% 64 2.53%Haloarcula marismortui 2642 268 10.14% 140 5.30% 95 3.60%
ATCC 43049Listeria innocua 2391 212 8.87% 278 11.63% 59 2.47%Methanocaldococcus jannaschil
DSN 2661 1427 106 7.43% 51 3.57% 14 0.98%Porphyromonas gingivalis W83 1233 78 6.38% 59 4.82% 40 3.27%Pseudomonas syringae pv.tomato 4177 458 10.96% 261 6.25% 180 4.31%
str.DC3000Ruminococcus obeum 2393 224 9.36% 245 10.24% 61 2.55%
ATCC 29174Silicibacter pomeroyi DSS 3399 5561 6.36% 204 6.00% 194 5.71%Sulfolobus solfataricus P2 2105 202 9.60% 128 6.08% 86 4.09%
Thermoplasma volcanium GSS1 1214 117 9.64% 89 7.33% 50 4.12%Vibrio parahaemolyticus 3259 346 9.80% 207 5.87% 123 3.49%
RIMD 2210633
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, 4GH77, 4
Crystal Structure Determination
Space group P21 Unit Cell (, ) a=68.86, b=124.97,
c=88.60, =107.7 MW Da (residue) 77083 (663) 1 Mol (AU) 2 SeMet (AU) 54 Wavelength() 0.97929 (peak) Resolution() 35.2-1.95 Number of unique reflections 104901 2 Redundancy 4.5 (4.0) 3 Completeness (%) 99.7 (97.3) 3 Rmerge (%) 16.0 (90.0) 3 I/ (I) 13.5 (1.4) 3
RCullis (anomalous) (%) 85 Figure of merit (%) 20.3
Resolution 35.2-1.95 Reflections (work/test) 99024/5209 Rcrystal/Rfree (%) 17.18/22.19 Rms deviation from ideal geometry Bond length ()/angle ()
0.015/1.486
No.of atoms (Protein/HETAT M) 11758/856 Mean B-value (2) (mainchain/sidechain)
12.77/15.15
Ramachandran plot statistic (%) Residues in most favored regions, in additional allowed regions, in generously allowed regions, in disallowed region
88.6 11.0 0.3 0.1
Data collection
Phasing
Refinement
X-ray Diffraction Data Collection and Processing:
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
Model Building:
HKL3000 program suite
3 cycles of Arp/warp model building: 1244 out 1332 residues built (93.4%).
sequence docked: 1211 residues.
Dimer Structure in Crystal and Solution
Calculated monomer molecular weight: 77.4kD, including vector derived residues.
Homologous Structures
8
Human intestinal maltase-glucoamylasePDB: 2QLYOverall sequence identity: 28%
Sulfolobus solfataricus -GlucosidasePDB:2G3MOverall sequence identity: 26%
Human Intestinal MGAM and SI
(1-4) high activity
(1-6)(1-4)
(1-4)
Maltase-Glucoamylase(MGAM)
Sucrase-Isomaltase(SI)
(exohydrolases)
amylose
amylopectin
Glucose
-Amylase(endohydrolase)
Catalytic Site
Catalytic domain
R.obeum -glucosidase: 366 a.a.Human NtMGAM: 362 a.a.
Structural alignment: 310 a.a. alignedRMSD: 1.68ÅSequence identity: 29.6%
Catalytic nucleophile: the residue D307 in magenta. Acid/base catalyst (possible): the residue D420 in green.
Substrate SpecificitySu
bstr
ate
Hyd
roly
zed
(mM
)
Maltose Sucrose Lactose
At least a maltase
Access to Catalytic Site
Glucoamylase ?
N- and C-terminal Domains
A Common Enzyme in Gut Microbiota
14
Glycosyl Hydrolases
(1-4) high activity
(1-6)(1-4)
(1-4)
Maltase-Glucoamylase(MGAM)
Sucrase-Isomaltase(SI)
(exohydrolases)
amylose
amylopectin
glucose
Conclusions
16
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 ? ……
1717
Acknowledgements ANL/MCSG A. Jochimiak H. An, G. Babnigg, L. Bigelow, A. Binkowski, C-s. Chang, S. Clancy, G. Cobb, M. Cuff, M. Donnelly, 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, J. Osipiuk, ,
G. Montelione, Ruthgers Univ. NESGCT. Terwilliger, Los Alamos, ITCSGZ. Derewenda, Univ. of Virginia, ITCSG Z. Dauter, NCIJ. Liang, Univ. of IllinoisD. Sherman, U. Michigan
Washington Univ.D. Fremont,T. Brett, C. Nelson,
Univ. of VirginiaW. Minor, M. Chruszcz, M. Cyborowski, M. Grabowski, P. Lasota, P. Miles,M. Zimmerman, H. Zheng
Univ. of Texas SWMC Z. Otwinowski, D. Borek, A. Kudlicki, A. Q. Mei, M. Rowicka
Northwestern Univ. W. Anderson, O. KiryukhinaD. Miller, G. Minasov, L. Shuvalova, X. Yang, Y. Tang
Univ. College London @ EBI, J. Thornton, C. Orengo, M. Bashton, R. Laskowski, D. Lee, R. Marsden, D. McKenzie, A. Todd, J. Watson
Univ. of Toronto A. Edwards, C. Arrowsmith, A. Savchenko,E. Evdokimova, J. Guthrie, A. Khachatryan, M. Kudrytska, T. Skarina, X. (Linda) Xu
Univ. of ChicagoO. Schneewind, D. Missiakas, P. Gornicki, S. Koide, ITCSGW-j. Tang,B. Roux,J. L. RobertsonM.R. Rosner,T. Kossiakoff, ITCSGV. Tereshko, Funding: NIH and DOE
M. Schiffer, A. SatherG. Shackelford,L. Stols, C. Tesar,R-y. Wu, L. Volkart, R-g. Zhang, M. Zhou,ANL/SBCN. Duke, S. Ginell,F. RotellaR. Wilton
Thank you