集胞藻PCC6803中N-乙酰鸟氨酸转氨酶的生化表征及结构分析

Biochemical Characterization and Structural Analysis of N-acetylornithine Transaminase from Synechocystis sp.PCC6803

旨在对集胞藻PCC6803中slr1022基因编码的N-乙酰鸟氨酸转氨酶进行生化表征及结构分析,为进一步研究该酶的催化功能及机制奠定理论基础。以集胞藻PCC6803基因组为模板,通过PCR扩增获得slr1022基因,将其连接到表达载体pET-28a上,转化大肠杆菌BL21(DE3)感受态。经IPTG诱导,Ni-NTA亲和层析纯化后获得重组Slr1022蛋白,然后通过紫外分光光度法对Slr1022蛋白的催化功能进行表征,并运用生物信息学软件对该蛋白进行结构分析。成功构建pET28a-slr1022重组表达质粒,并诱导表达重组Slr1022蛋白,SDS-PAGE电泳鉴定该蛋白分子量约为50 kD,与理论大小相符。Slr1022蛋白与底物N-乙酰鸟氨酸的结合常数K_m和最大反应速度V_(max)分别是0.12 mmol/L和0.60 μmol/(L·s),并且Slr1022蛋白与另一底物α-酮戊二酸的K_(m)和V_(max)分别是0.039 mmol/L和0.65 μmol/(L·s)。Slr1022蛋白在pH 8.5时催化活性最强。Slr1022蛋白和其他来源的N-乙酰鸟氨酸转氨酶氨基酸序列有一定的同源性,而且活性位点的氨基酸残基高度保守。成功克隆并表达纯化出Slr1022蛋白,酶学性质和生物信息学研究表明Slr1022蛋白为N-乙酰鸟氨酸转氨酶。

The aim of this study is to characterize the biochemical and structural properties of N-acetylornithine transaminase encoded by slr1022 gene from Synechocystis sp.PCC6803,for laying theoretical foundation in further study on the catalytic function and mechanism of the enzyme.The slr1022 gene was obtained through PCR amplification using the genomic DNA of Synechocystis sp.PCC6803 as template,and then was linked to the expression vector pET-28a.The constructed plasmid was transformed into Escherichia coli strain BL21(DE3).The recombinant Slr1022 protein was purified by Ni-NTA affinity chromatography after the expressing strain was induced by IPTG.The catalytic function of the Slr1022 protein was characterized preliminarily through UV spectrophotometric methods,and the structure of the Slr1022 protein was analyzed by bioinformatics software.The recombinant expressing plasmid of the pET28a-slr1022 was constructed and the Slr1022 protein was expressed successfully.The molecular weight of the protein by SDS-PAGE was about 50 kD,which was consistent with the theoretical size.The binding constant Km and maximum reaction velocity V_(max) of the Slr1022 protein with substrate N-acetylornithine were 0.12 mmol/L and 0.60μmol/(L·s),respectively,and the K_(m) and V_(max) of Slr1022 protein withα-ketoglutarate were 0.039 mmol/L and 0.65μmol/(L·s),respectively.Slr1022 protein had the highest catalytic activity at pH 8.5.The amino acid sequence of Slr1022 protein and other N-acetylornithine transaminase from different sources shared certain homology,and the amino acid residues of the active site were highly conserved.In conclusion,the slr1022 gene is successfully cloned and the Slr1022 protein is expressed and purified,enzymatic properties and bioinformatics analysis demonstrates that Slr1022 protein is N-acetylornithine transaminase.