title: Structural and kinetic dissection of the endo-α-1,2-mannanase activity of bacterial GH99 glycoside hydrolases from Bacteroides spp.
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 (Glc1-3 Man9 GlcNAc2 ), 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 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.
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010 (Australia).
authors.forename: Zalihe
authors.initials: Z
authors.lastname: Hakki
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thompson
authors.forename: Stephanie
authors.initials: S
authors.lastname: Bellmaine
authors.forename: Gaetano
authors.initials: G
authors.lastname: Speciale
authors.forename: Gideon J
authors.initials: GJ
authors.lastname: Davies
authors.forename: Spencer J
authors.initials: SJ
authors.lastname: Williams
mesh_terms: D001439:Bacteroides / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006867:Hydrolases / Q000378:metabolism*; D008361:Mannosidases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular
chemical_list: D006867:Hydrolases; D006026:Glycoside Hydrolases; D008361:Mannosidases
keywords: carbohydrates; endomannosidase; enzymes; mechanism; structural biology