Structural Insights Into The Catalytic Mechanism Of GH Family 46 Chitosanases

Chitooligosaccharides have a variety of unique physiological functions,which is strongly related to the degree of polymerization and the degree of acetylation.Chitosanase can specifically degrade chitosan to produce chitooligosaccharides,with high catalytic efficiency,easy product separation,mild reaction conditions,and industrial application value.Most of the reported chitosanases follow the endo-type reaction mechanism,which leads to random degree of polymerization and complex acetylation sites in the hydrolysates.The end products of the hydrolysis are mostly short-chain products,such as chitobiose and chitotriose.Therefore,it is of great significance to understand the molecular mechanism of difference in degree of polymerization of chitosanase products,and to explore the preparation of chitooligosaccharides by chitosanase in a specific range of polymerization degree.The purpose of this study is to analyze the complex structure of GH family 46 chitosanases by X-ray crystallography,understand the substrate binding and catalytic mechanism of the GH family 46 chitosanases,and explore the molecular mechanism of difference in degree of polymerization of chitosanase hydrolysates.Firstly,complex structures of two GH46 chitosanases with chitotetraose and chitopentose,GsCsn46A derived from Gynuella sunshinyii and BaCsn46A derived from Bacillus amyloliquefaciens were analyzed by X-ray crystallography,respectively.The detailed substrate binding mechanism of GH family 46 chitosanase and chitooligosaccharides(-5—+3 sites)was elucidated,and three new subsites were found in the non-reducing ends of the GH family 46 chitosanase catalytic groove.Secondly,the complex structures show that the sugar chain of substrate in the catalytic groove of GsCsn46A is bound to the non-reducing ends,and the substrate binding cleft of GsCsn46A is in the "closed" state.This phenomenon validates the allosteric reaction mechanism of the GH family 46 chitosanase in the catalytic process:the sugar chain substrate is preferentially recognized and bound in the non-reducing ends,and the catalytic groove undergoes allosteric reaction.The substrate binding cleft "closes" and an inverting hydrolysis reaction occurs.This discovery further improves the existing substrate recognition and catalytic mechanism of the GH family 46 chitosanases.Finally,based on the obtained structural information of the two chitosanases,16 sets of site-directed mutations were designed.By measuring the enzymatic activity of each mutant towards chitosan and the distribution of hydrolysates,the function of key amino acid residues in the catalytic groove of GH46 family chitosanase was verified,and a mutant N21W of GsCsn46A was screened out.This mutant only retained a certain enzymatic activity towards chitosan and almost lost the ability to hydrolyze chitotetraose,so that the proportion of chitooligosaccharides with degree of polymerization>4 in the hydrolysates was significantly increased,which could be used to prepare a series of chitooligosaccharides with higher degree of polymerization.In this study,the complex structures of the two GH family 46 chitosanases were analyzed,the substrate binding and catalytic mechanism of the GH family 46 chitosanase was clarified,and the potential mechanism of difference in the degree of polymerization of GH family 46 chitosanase products was revealed at the molecular level.On this basis,according to the protein structure information,a chitosanase mutant producing more chitooligosaccharides with higher degree of polymerization in the hydrolysates was obtained through site-directed mutagenesis.The above research provides a theoretical basis for the high-efficient enzymatic preparation of chitooligosaccharides with specific degree of polymerization,and also provides a research basis for further rational protein engineering of chitosanases.