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Structural and kinetic dissection of the endo-α-1,2-mannanase activity of bacterial GH99 glycoside hydrolases from Bacteroides spp

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JournalChemistry : A European Journal
DateE-pub ahead of print - 8 Dec 2014
DatePublished (current) - 26 Jan 2015
Issue number5
Volume21
Number of pages12
Pages (from-to)1966-1977
Early online date8/12/14
Original languageEnglish

Abstract

Glycoside hydrolase family 99 (GH99) was created to categorize sequence-related glycosidases possessing endo -a-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-αMan→1,2-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 wildtype or inactive mutants, respectively, of B. xylanisolvens GH99 reveal the structural basis for binding to d-mannoserather than d-glucose-configured substrates.

    Research areas

  • Carbohydrates, Endomannosidase, Enzymes, Mechanism, Structural biology

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