链霉菌来源谷氨酰胺合成酶GLnA_Shy的性质表征  

Characterization of glutamine synthetase GLnA_Shy from Streptomyces

作　　者：刘辉 刘璋敏 冯雁[1] 李谦[2] 崔莉[1] LIU Hui;LIU Zhangmin;FENG Yan;LI Qian;CUI Li(State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiaotong University,Shanghai 200240,China;School of Life Sciences and Technology,China Pharmaceutical University,Nanjing 210009,China)

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点实验室,上海200240 [2]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《南京工业大学学报（自然科学版）》2020年第2期249-256,共8页Journal of Nanjing Tech University(Natural Science Edition)

基　　金：国家自然科学基金(31770098,31620103901)。

摘　　要：在大肠杆菌BL21(DE3)中对吸水链霉菌5008的GLnyShy基因进行克隆表达。GLnAShy蛋白由469个氨基酸组成,其分子量约为5.2×10^4。体外酶学性质及酶动力学测定结果显示,GLnAShy酶催化反应的最适温度为35℃,最适pH为7.5,对底物谷氨酸的米氏常数(Km)和催化常数(kcat)分别为(21.8±3.2)μmol/L和(3.8±0.5)×10^-3 s^-1。进化树分析和同源建模结果显示GLnAShy蛋白属于谷氨酰胺合成酶(GS)家族,该酶包含一个较小的N端结构域(残基区域为16~94)和一个较大的C端结构域(残基区域为101~466),其三级结构具有GS典型的双通道结构,与底物谷氨酸的结合位点为Glu131、Ser264、Arg320和Arg359。谷氨酰胺合成酶在含氮化合物的生产上应用广泛,通过解析GLnAShy的功能,为进一步对其进行定点改造提供了理论基础。GLnAShy protein contains 469 amino acids with a molecular weight of 5.2×104. The gene GLnAShy from Streptomyces hygroscopicus 5008 was expressed in Escherichia coli BL21(DE3) which encoded protein GLnAShy. Results of enzymatic kinetics showed that the optimum temperature and pH of GLnAShy protein catalyzing the conversion of glutamate to glutamine were 35 ℃ and 7.5, respectively. The michaelis constant(Km) and catalytic rate constant(kcat) of GLnAShy were(21.8±3.2) μmol/L and(3.8±0.5)×10^-3 s^-1, respectively. The results of phylogenetic analysis and homology modeling indicated that GLnAShy protein belonged to glutamine synthase(GS) family. It had a smaller N-terminal domain(amino acid residues from 16 to 94) and a larger C-terminal domain(amino acid residues from 101 to 466), and had a classical bifunnel structure of GS. The predicted key amino acids of its binding sites with glutamate were Glu131, Ser264, Arg320 and Arg359. The functional analyses of GLnAShy offered a foundation for further site-specific protein modification.

关 键 词：谷氨酰胺合成酶 大肠杆菌 同源建模 链霉菌 克隆表达 

分 类 号：Q814[生物学—生物工程]