Three-dimensional Structure Determination And Catalytic Mechanism Analysis Of Bacterial α-glucosidase HaG

α-Glucosidase is one of the most important enzymes that take part in the primary metabolism, and thus can be found in varieties of organisms. It is related to some kinds of diseases such as diabetes and cancer, and has been widely applied in medicine, and industrial production. In this thesis, the crystal structure of α-glucosidase Ha G from marine halophilic bacterial was determined by X-Ray crystallography. Structural analysis combining with biochemical experiments explained the substrate specificity and catalytic mechanism of Ha G, revealed the relationship between structure and function, which paves the way to reconstruct and further utilize α-glucosidases. The main content and results are as follows:1. Overexpression, purification, crystallization and structure determinatino of wild type Ha G protein. The gene of Ha G protein was cloned into p FLAG-CTS vector and overexpressed in E. coli. The active target protein was extracted from cells by osmotic shock method, and then highly purified by weak Anion-exchange Chromatography and Size-exclusion Chromatography for crystallization.Single crystal with high quality was obtained by sitting vapor method after screening and optimizing the crystallization conditions. The diffraction data was collected from Synchrotron Radiation Center in Japan, and the structure of Ha G was then solved by molecular replacement. It is shown that Ha G structure consists of a catalytic N domain, a C domain that is important for maintaining the structure stability, and a subdomain. The active site pocket of Ha G is defined by 15 residues, which are located in the middle of the(α/β)8 barrel.2. Structure determination of Ha G and its mutants in complex with different ligands. Inactive mutant E271 Q, D202 N and D333 N were obtained by site-directed mutagenesis. Four complex crystals were obtained by co-crystallization or soaking:(1) Glucosyl-Ha G, which is the reaction intermediate of wild type Ha G, represents a real process during the catalytic reaction of α-glucosidase;(2) E271Q-Mal is the first α-(1,4) glucosidic bond substrate complex in GH13 family. There are 114 residues whose electronic density was invisible in E271Q-Mal, which are supposed to be flexible during the reaction, makes it easier for substrate to get into the active site pocket;(3) D202N-Glu-Gly, which clarified the reaction characteristics of producing αGG, simulated the transglucosylation of Ha G, by using glycerol as acceptor;(4) D333N-Rb showed the position of Rb+ by collecting the anomalous signal of Rb+ under specific-wavelength X-ray.3. The structural basis of substrate specificity and catalytic mechanism. Based on the comparison of Ha G complexes with other enzymes, combined with mutagenesis and enzymatic experiments, the substrate specificity were revealed.(1) The disaccharide-specificity of Ha G is caused by steric hindrance provided by long βâ†'α loop 4, which covers most part of the entrance.(2) Two residues, Thr203 and Phe297, were found to decide the glycosidic linkage recognition, which was also assisted by residue Gly228. Structure analysis of all the Ha G complexes made some new insights into the catalytic mechanism of α-glucosidase and provide structural basis for classic double-displacement mechanism.