TY - JOUR
T1 - Structural and kinetic dissection of the endo-α-1,2-mannanase activity of bacterial GH99 glycoside hydrolases from Bacteroides spp
AU - Hakki, Zalihe
AU - Thompson, Andrew J.
AU - Bellmaine, Stephanie
AU - Speciale, Gaetano
AU - Davies, Gideon J.
AU - Williams, Spencer J.
PY - 2015/1/26
Y1 - 2015/1/26
N2 - 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.
AB - 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.
KW - Carbohydrates
KW - Endomannosidase
KW - Enzymes
KW - Mechanism
KW - Structural biology
UR - http://www.scopus.com/inward/record.url?scp=84921378183&partnerID=8YFLogxK
U2 - 10.1002/chem.201405539
DO - 10.1002/chem.201405539
M3 - Article
AN - SCOPUS:84921378183
SN - 0947-6539
VL - 21
SP - 1966
EP - 1977
JO - Chemistry : A European Journal
JF - Chemistry : A European Journal
IS - 5
ER -