Kemin Tan and Andrzej Joachimiak

2009 NIGMS Workshop: Enabling Technologies for Structural Biology

March 4-6, 2009

The Structure and Function of -Glucosidase from Human Gut Bacterium

Ruminococcus Obeum

Gut Microbiota

Division

Actinobacteria

Bacterioidetes

Firmicutes

Proteobacteria…etc.

{

Genus

Clostridium

EubacteriumRuminococcus…etc.

{Species

Gnavusobeum…

etc.

Strain

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 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 SpecificityS

ub

str

ate

Hy

dro

lyze

d (

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