Structural And Functional Analysis Of Akkermansia Muciniphila ?-N-acetylhexosaminidases

Akkermansia Muciniphila,a gram-negative bacterium,is widely existence in eukaryote,especially with an abundance of about 1%-5%in the human intestine.Akkermansia Muciniphila may exhibit potential responses and immune responses by communicating with the host,promoting intestinal barrier protection mechanism,and regulating the intestinal microbial bacteria,which in return playing a vital role in the host microecological stability.Akkermansia Muciniphila could degrade mucin which as a sole carbon source into N-acetylglucosamine,N-acetylgalactosamine,sulfate,etc.and provide basic energy for bacterial survival by expressing and secreting?-N-acetylhexosaminidases,a kind of glycosidase.In this study,we determined two?-N-acetylhexosaminidases by analyzing the Akkermansia Muciniphila genes with combining the bioinformatic strategy.The two?-N-acetylhexosaminidase genes were named Am2301 and Am2136,respectively,according to their gene and uniport numbers.The Am2301 and Am2136 were expressed in E.coli system,and then purified by nickel column and gel filtration chromatography successfully.Finally,the proteins with high purity and good homogeneity which were determined by SDS-PAGE and SEC results were suitable for enzymatic characteristics and crystallography studies.The substrate specificity was determined by using four different substrates,p NP-?-Gal NAc,pNP-?-GlcNAc p NP-?-Gal NAc,and p NP-?-Glc NAc.The results showed that the recombinant proteins Am2301 and Am2136 all had the highest catalytic efficiency under?linked glucosamine,while the catalytic efficieny could not be detected when the substrate linked with?bond.These results indicated that the recombinant proteins Am2301and Am2136 were able to specifically remove?-linked N-acetylhexosylamine.For Am2301,the optimized p H and temperature was 7.0 and 37°C,respectively.With the presence of appropriate amount of Zn²⁺,the enzymatic activity increased while Mg²⁺,Ca²⁺and Mn²⁺inhibited the enzymatic activity.However,the optimal p H of Am2136 was 7.5,and it presented excellent catalytic activity at a broad temperature from 25 to 40°C.Unlike Am2301,its enzymatic activity decreased after adding a certain amount of Ca²⁺,Mn²⁺,and Zn²⁺,especially after adding Zn ions,Am2136 was completely inactivated.Then we determined the enzymatic characteristics of Am2301 and Am2136 at the optimal substrate of pNP-?-GlcNAc with catalytic sites confirmation by combining with mutation analysis.The enzymatic efficiency of mutated species decreased significantly when compared with the wild-type species.For Am2301,the enzymatic efficiency of D278A,E279A and Y373F species decreased about 2600 times,3500 times and 1226 times separately.For Am2136,the enzymatic efficiency of D412A and E413A species decreased about 4297times and 210 times separately.The results indicated that there are two conserved amino acids,aspartic acid and glutamic acid,which located in the binding pocket,may play an important role in the catalytic process.Subsequently,we screened and optimized the crystallization conditions of Am2301 and Am2136 at each different protein concentration,respectively,and finally obtained high quality crystals and collected high-resolution data at the resolution of 1.80(?)and 1.60(?),respectively.For structure determination,since the sequence similarity of Am2301 with published structure was about 35%and molecular replacement method could be performed for phasing determination and selenomethionine method was used to solve the phasing issue of Am2136 and finally obtained the structure.Finally,we analyzed the apo and complex structures of Am2301 and Am2136 to investigate the structural characteristics and catalytic mechanism of?-N-acetylhexosaminidases in Akkermansia Muciniphila.The results showed that the catalytic domain of Akkermansia Muciniphila?-N-acetylhexosaminidases was the classical(?/?)₈ TIM barrel domain,and their substrate binding pocket was located in the TIM barrel center.The complex structures of Am2301 and Am2136 with Glc NAc indicated that Glc NAc could be stabilized via various interactions among few crucial amino acid residues near the binding pocket.The active oxygen of the C-2 acetyl amino group of the substrate acted as a catalytic nucleophile,through attacking the catalytic active site amino acids,thus forming a covalently linked glycosidase,promoting the substrate to form an oxazoline intermediate.That is so called substrate-assisted catalytic mechanism.With SWISS-MODEL server,the three-dimensional structures of the other nine?-N-acetylhexosaminidases from Akkermansia Muciniphila were predicted and structurally alignment had been done.The results showed that the catalytic domains were extraordinarily conserved,which played a very important role in maintaining their enzymatic properties for?-N-acetylhexosaminidases.In summary,the analysis of?-N-acetylhexosaminidases from Akkermansia Muciniphila through structural biological strategy,was the first crystal structure of key proteins of Akkermansia Muciniphila,which is important for further investigation of the role of?-N-acetylhexosaminidases in maintaining Akkermansia Muciniphila growth and reproduction,promoting intestinal barrier protection mechanisms,regulating microbial bacteria in the intestine and possibly maintaining micro-ecological stability.