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Structural and Kinetic Dissection of the endo-alpha-1,2-Mannanase Activity of Bacterial GH99 Glycoside Hydrolases from Bacteroides spp.

DOI: 10.1002/chem.201405539 DOI Help
PMID: 25487964 PMID Help

Authors: Zalihe Hakki (University of Melbourne) , Andrew J. Thompson (University of York) , Stephanie Bellmaine (University of Melbourne) , Gaetano Speciale (University of Melbourne) , Gideon J. Davies (University of York) , Spencer J. Williams (University of Melbourne)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Chemistry - A European Journal , VOL 21 (5) , PAGES 1966 - 1977

State: Published (Approved)
Published: January 2015
Diamond Proposal Number(s): 7864

Abstract: Glycoside hydrolase family 99 (GH99) was created to categorize sequence-related glycosidases possessing endo-α-mannosidase activity: the cleavage of mannosidic linkages within eukaryotic N-glycan precursors (Glc1–3Man9GlcNAc2), releasing mono-, di- and triglucosylated-mannose (Glc1–3-1,3-Man). GH99 family members have recently been implicated in the ability of Bacteroides spp., present within the gut microbiota, to metabolize fungal cell wall α-mannans, releasing α-1,3-mannobiose by hydrolysing αMan-1,3-αMan1,2-αMan-1,2-αMan sequences within branches off the main α-1,6-mannan backbone. We report the development of a series of substrates and inhibitors, which we use to kinetically and structurally characterise this novel endo-α-1,2-mannanase activity of bacterial GH99 enzymes from Bacteroides thetaiotaomicron and xylanisolvens. These data reveal an approximate 5 kJ mol−1 preference for mannose-configured substrates in the −2 subsite (relative to glucose), which inspired the development of a new inhibitor, α-mannopyranosyl-1,3-isofagomine (ManIFG), the most potent (bacterial) GH99 inhibitor reported to date. X-ray structures of ManIFG or a substrate in complex with wild-type or inactive mutants, respectively, of B. xylanisolvens GH99 reveal the structural basis for binding to D-mannose- rather than D-glucose-configured substrates.

Journal Keywords: Carbohydrates;Endomannosidase;Enzymes;Mechanism;Structural Biology

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography

Added On: 26/03/2015 16:46

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