Study On The Structure And Function Of β-N-acetylhexosaminidase Am0868 From Akkermansia Muciniphila

Akkermansia muciniphlia,a mucin-degrading bacterium colonizing in the mucus layer,is known to have an important value on improving the intestinal barrier and effective immune responses in human intestine.In whole-genome analyses of A.muciniphila,twelve putative glycosidase candidate genes have been identified to encodeβ-N-acetylhexosaminidases,and predominantly represented in the family GH20.β-N-acetylhexosaminidases are found to be capable of cleaving the specific glycoside linkages and play a key role in the process of mucin degradation,and their enzymatic characteristics and catalytic mechanism are currently widely studied.In this study,A.muciniphilaβ-N-acetylhexosaminidase（Am0868）recombinant protein was obtained by the heterogenic expression system of E.coli.The crystal structure of Am0868and Am0868 complexed with the small molecule,Glc NAc were solved by X-ray crystallography,the substrate specificity and biochemical properties of Am0868 was explored in vitro,the catalytic effects of aspartic acid（D326）and glutamic acid（E327）were verified by constructing the Am0868 D326A and Am0868 E327A mutants and analyzing kinetics of the two mutant enzymes.The structure analysis showed that the overall structure of the Am0868 consists of an N-terminal domain and a C-terminal catalytic domain.The catalytic domain is folded as a typical（β/α）₈barrel（TIM barrel）,where the active site resides.The small molecule substrate Glc NAc binds to the catalytic active center through hydrophobic and hydrogen bond interactions.Trp376,Trp393 and Trp474 form a hydrophobic pocket to help stabilize the substrateβ-Glc NAc in the active center.Asp326 and Tyr420 appear important for correctly positioning of the 2-acetylamino group of the substrate,while Glu327 plays a role in acid-base catalysis.The results of the substrate specificity determination showed that Am0868 exhibited high preference forβ-Glc NAc.Am0868 did not show catalytic activity towards substrates lacking an N-acetamido group,indicating that the 2-acetamido group of the substrate was indispensable for catalysis.The results of the biochemical characteristics study revealed that Am0868 had broad p H tolerance and good thermal stability,but the activity of Am0868 was reduced significantly by divalent metal ions Zn²⁺,Cu²⁺,Cd²⁺and Ni²⁺.Kinetic analysis showed that,compared with the wild type,the catalytic efficiency of mutant Am0868 D326A is almost completely lost,while the E327A mutant still retained partial enzyme activity.The results of crystallographic and mutational studies confirmed that Asp326 and Glu327 are the key catalytic residues of Am0868.Our results reveal the key role of aspartic acid in the substrate-assisted catalytic mechanism of A.muciniphilaβ-N-acetylhexosaminidases Am0868,and provide detailed biochemical information for the study of the potential of Am0868 in vivo mucin hydrolysis.