Study of Regulatory Mechanism of Catalysis by Cobalamin-dependent Enzymes

1.甘油脱氢酶的X-射线衍射分析对重组肺炎克雷伯氏菌甘油脱氢酶进行了纯化和表征。在2.1 A分辨率下测定了甘油脱氢酶与氰钴胺和丙二醇复合物的晶体结构。所阐明的结构特征与二醇脱氢酶的结构特征非常相似。尽管外消旋丙烷-1，2-二醇用于结晶，但与甘油脱氢酶结合的底物被分配给（R）-异构体。二醇脱氢酶活性位点残基的突变分析为了研究这些残基的作用，构建并分析了这些残基的丙氨酸突变体。结果 关于我们 表明H143和E170残基在催化过程中的重要性。通过构建H143 E、H143 K、H143 Q和H143 L突变体进一步研究了残基H143的作用。结果表明，组氨酸与丙二醇的2-羟基之间的氢键对催化反应的重要性。结果表明，Sα224对活化C_2Cbl的Co-C键和防止催化失活具有重要作用。3.大鼠甲硫氨酸合成酶H773 G突变体的构建为了构建与甲钴胺以碱基对方式结合的突变型甲硫氨酸合成酶，采用定点突变的方法，将His 773突变为Gly。利用杆状病毒表达系统在昆虫细胞中高效表达大鼠甲硫氨酸合酶，试图获得突变型甲硫氨酸合酶，但突变蛋白表达失败。少

1.X-ray analysis of glycerol dehydratase.Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity and characterized. The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The structural features elucidated are quite similar to those of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.2.Mutational analysis of active-site residues of diol dehydratase.X-ray structure of diol dehydratase showed the seven amino acid residues interacting directly with active site K^+ and/or hydroxy group of substrate. To investigate the roles of these residues, alanine mutants of these residues were constructed and analyzed. The results … More indicated the importance of H143 and E170 residues in the catalytic process. Role of the residue, H143, was examined further by constructing H143E,H143K,H143Q, and H 143L mutants. The results indicated the importance of hydrogen bond between His and 2-hydroxy group of propanediol for the catalytic reaction.Role of two cobalamin-binding residues was investigated with site-directed mutagenesis. The results indicated that Sα224 is important for the activation of the Co-C bond of AdoCbl and for the prevention from inactivation during catalysis. The importance of Kβ135 for the binding of AdoCbl was also suggested.3.Construction of H773G mutant of rat methionine synthase.To construct mutant methionine synthase to which methylcobalamin was bound in base-on mode, His773 to Gly mutation was introduced into methionine synthase gene by site-directed mutagenesis. The method of high-level expression of rat methionine synthase in insect cells using a baculovirus expression system was tried to get the mutant methionine synthase, however, expression of mutant protein failed. Less