青枯雷尔氏菌GMI1000菌株龙胆酸1,2-双加氧酶活性中心关键氨基酸残基的鉴定(英文)

Identification of Critical Residues for Activity of a Gentisate 1,2-dioxygenase from Ralstonia solanacearum GMI1000

生物信息学分析表明,位于青枯雷尔氏菌GMI1000菌株的染色体上的读码框RSc1087可能编码一个龙胆酸1,2-双加氧酶.本研究克隆、表达了该基因,并通过亲和层析对该基因表达产物进行了纯化.酶学测试结果证实,该基因编码的正是龙胆酸1,2-双加氧酶.SDS-PAGE结果表明,该酶亚基分子量约为38×103.基因的定点突变揭示105位、107位和146位组氨酸残基是该酶活性中心的关键氨基酸残基.

Locus RSc1087 of the chromosome from a soil - borne Gram-negtive bacterium, Ralstonia solanacearttm GMI1000 was putative to encode a gentisate 1,2-dioxygenase by the analysis of bioinformatics. It was cloned and overexpressed in Escherichia coll. The resulting product incorporated a （His） 6 tag was purified to homogeneity from the harvested cell extracts by affinity chromatograhpy. Enzyme assays confirmed that RSc1087 encoded a gentisate 1,2-dioxygenase. SDS-PAGE showed that the polypeptide exhibited an approximate molecular mass of 38 × 10^3. Site-directed mutagenesis revealed that Hisl05, Hisl07 and His146 were the crucial residues involved in the catalytic activity of gentisate 1,2-dioxygenase R. solanacearum GMII000.