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- Title
Structural and Kinetic Dissection of the endo-α-1,2-Mannanase Activity of Bacterial GH99 Glycoside Hydrolases from Bacteroides spp.
- Authors
Hakki, Zalihe; Thompson, Andrew J.; Bellmaine, Stephanie; Speciale, Gaetano; Davies, Gideon J.; Williams, Spencer J.
- 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-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.
- Subjects
ENZYMES; CHROMATOGRAPHIC analysis; MANNOSE; YEAST; DISACCHARIDES; HYDROLASES
- Publication
Chemistry - A European Journal, 2015, Vol 21, Issue 5, p1966
- ISSN
0947-6539
- Publication type
Article
- DOI
10.1002/chem.201405539