吸水链霉菌5008中谷氨酸脱氢酶SHJG＿7666的性质表征  

Characterization of glutamate dehydrogenase SHJG＿7666 from Streptomyces hygroscopicus 5008

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

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,南京210009

出　　处：《中国药科大学学报》2018年第3期360-368,共9页Journal of China Pharmaceutical University

基　　金：国家自然科学基金资助项目(No.31620103901;No.31770098)~~

摘　　要：谷氨酸脱氢酶可在NAD(P)H辅因子协助下催化α-酮戊二酸和谷氨酸之间的转化,是氮代谢途径的关键酶。基于基因组BLAST分析预测,吸水链霉菌5008(S5008)中SHJG_7666是谷氨酸脱氢酶的编码基因。本研究在大肠杆菌中对SHJG_7666蛋白进行了异源表达,并对其酶学性质进行了表征。系统发育树及同源建模结构分析显示SHJG_7666具有保守的谷氨酸、α-酮戊二酸结合域以及经典的GXGXXG二核苷酸结合基序,其活性中心由Lys^(60),Lys^(78),Asp^(120)催化功能位点及Ser^(36),Gly^(38),Gln^(119),Asp^(166),Asn^(300),Ala^(330)谷氨酸、α-酮戊二酸结合位点组成,属于Glu/Leu/Phe/Val脱氢酶家族;体外生化实验显示,其催化α-酮戊二酸转化为谷氨酸的最适p H和温度分别为7.5和37℃,对底物α-酮戊二酸K_m为(25.3±9.1)μmol/L,k_(cat)为(3±0.8)×10^(-5)s^(-1)。本研究验证了SHJG_7666谷氨酸脱氢酶的催化功能,揭示了其活性中心关键氨基酸残基,为利用蛋白质工程手段对SHJG_7666进行分子设计以提高其催化活力,进一步利用代谢工程手段对宿主氮代谢进行调控奠定了基础。Glutamate dehydrogenase（GDH）a key enzyme in the nitrogen metabolism pathway catalyzes the conversion betweenα-ketoglutarate and glutamate reversibly using NAD（P）H as a cofactor.Based on genomic studies,it was concluded that SHJG7666 was a potential GDH in Streptomyces hygroscopicus 5008（S5008）,and its expression level in vivo was positively correlated with the biosynthesis of an important aminocyclol compound validamycin.Phylogenetic tree analysis showed that the S5008 SHJG7666 GDH belonged to the Glu/Leu/Phe/Val dehydrogenase family,with conserved glutamate-α-ketoglutarate binding domain and the classical GXGXXG dinucleotide binding motif.Further homologous modeling and structural comparison revealed that SHJG7666 contained conserved Lys^60,Lys^78and Asp^120catalytic functional sites and ligand binding residues Ser^36,Gly^38,Gln^（119）and Asp^166,Asn^300,Ala^330.Moreover,recombinant expression of SHJG7666 in E.coli and in vitro enzyme activity demonstrated that glutamate dehydrogenase can convert ammonium salt to glutamate with p H and temperaturebeing optimal at 7.5 and 37°C respectively.Enzyme activity under optimum reaction condition has Kmvalue of（25.3±9.1）μmol/L and kcatof（3±0.8）×10^-5s^-1for the substrateα-ketoglutarate.Results of this study further improved the catalytic activity of SHJG7666,thus laying the foundation for the ultimate increase of validamycin production.

关 键 词：谷氨酸脱氢酶 氮代谢 井冈霉素 同源建模 酶学性质表征 

分 类 号：Q814[生物学—生物工程]