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J. Biol. Chem., Vol. 282, Issue 15, 11291-11299, April 13, 2007

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Mannose Foraging by Bacteroides thetaiotaomicron

STRUCTURE AND SPECIFICITY OF THE -MANNOSIDASE, BtMan2A^*

Louise E. Tailford¹, Victoria A. Money¹, Nicola L. Smith¹, Claire Dumon, Gideon J. Davies, and Harry J. Gilbert²

From the Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH and the Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, United Kingdom

The human colonic bacterium Bacteroides thetaiotaomicron, which plays an important role in maintaining human health, produces an extensive array of exo-acting glycoside hydrolases (GH), including 32 family GH2 glycoside hydrolases. Although it is likely that these enzymes enable the organism to utilize dietary and host glycans as major nutrient sources, the biochemical properties of these GH2 glycoside hydrolases are currently unclear. Here we report the biochemical properties and crystal structure of the GH2 B. thetaiotaomicron enzyme BtMan2A. Kinetic analysis demonstrates that BtMan2A is a -mannosidase in which substrate binding energy is provided principally by the glycone binding site, whereas aglycone recognition is highly plastic. The three-dimensional structure, determined to a resolution of 1.7Å_, reveals a five-domain structure that is globally similar to the Escherichia coli LacZ -galactosidase. The catalytic center is housed mainly within a (/)₈ barrel although the N-terminal domain also contributes to the active site topology. The nature of the substrate-binding residues is quite distinct from other GH2 enzymes of known structure, instead they are similar to other clan GH-A enzymes specific for manno-configured substrates. Mutagenesis studies, informed by the crystal structure, identified a WDW motif in the N-terminal domain that makes a significant contribution to catalytic activity. The observation that this motif is invariant in GH2 mannosidases points to a generic role for these residues in this enzyme class. The identification of GH-A clan and GH2 specific residues in the active site of BtMan2A explains why this enzyme is able to harness substrate binding at the proximal glycone binding site more efficiently than mannan-hydrolyzing glycoside hydrolases in related enzyme families. The catalytic properties of BtMan2A are consistent with the flexible nutrient acquisition displayed by the colonic bacterium.

Received for publication, November 28, 2006 , and in revised form, January 29, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ These authors contributed equally to the results of this work.

² To whom correspondence should be addressed. E-mail: h.j.gilbert{at}ncl.ac.uk.

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