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吸水链霉菌5008中谷氨酸脱氢酶SHJG_7666的性质表征

刘璋敏, 巢佳佳, 冯雁, 李谦, 崔莉

刘璋敏, 巢佳佳, 冯雁, 李谦, 崔莉. 吸水链霉菌5008中谷氨酸脱氢酶SHJG_7666的性质表征[J]. 中国药科大学学报, 2018, 49(3): 360-368. DOI: 10.11665/j.issn.1000-5048.20180316
引用本文: 刘璋敏, 巢佳佳, 冯雁, 李谦, 崔莉. 吸水链霉菌5008中谷氨酸脱氢酶SHJG_7666的性质表征[J]. 中国药科大学学报, 2018, 49(3): 360-368. DOI: 10.11665/j.issn.1000-5048.20180316
LIU Zhangmin, CHAO Jiajia, FENG Yan, LI Qian, CUI Li. Characterization of glutamate dehydrogenase SHJG_7666 from Streptomyces hygroscopicus 5008[J]. Journal of China Pharmaceutical University, 2018, 49(3): 360-368. DOI: 10.11665/j.issn.1000-5048.20180316
Citation: LIU Zhangmin, CHAO Jiajia, FENG Yan, LI Qian, CUI Li. Characterization of glutamate dehydrogenase SHJG_7666 from Streptomyces hygroscopicus 5008[J]. Journal of China Pharmaceutical University, 2018, 49(3): 360-368. DOI: 10.11665/j.issn.1000-5048.20180316

吸水链霉菌5008中谷氨酸脱氢酶SHJG_7666的性质表征

基金项目: 国家自然科学基金资助项目(No.31620103901,No.31770098)

Characterization of glutamate dehydrogenase SHJG_7666 from Streptomyces hygroscopicus 5008

  • 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Lys60,Lys78,Asp120催化功能位点及Ser36,Gly38,Gln119,Asp166,Asn300,Ala330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pH和温度分别为7.5和37 ℃,对底物α-酮戊二酸Km为(25.3±9.1)μmol/L,kcat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。
    Abstract: Glutamate dehydrogenase(GDH)a key enzyme in the nitrogen metabolism pathway catalyzes the conversion between α-ketoglutarate and glutamate reversibly using NAD(P)H as a cofactor. Based on genomic studies, it was concluded that SHJG_7666 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 SHJG_7666 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 SHJG_7666 contained conserved Lys60, Lys78 and Asp120 catalytic functional sites and ligand binding residues Ser36, Gly38, Gln119and Asp166, Asn300, Ala330. Moreover, recombinant expression of SHJG_7666 in E. coli and in vitro enzyme activity demonstrated that glutamate dehydrogenase can convert ammonium salt to glutamate with pH and temperature being optimal at 7. 5 and 37 °C respectively. Enzyme activity under optimum reaction condition has Km value of(25. 3±9. 1)μmol/L and kcat of(3±0. 8)×10-5 s-1 for the substrate α-ketoglutarate. Results of this study further improved the catalytic activity of SHJG_7666, thus laying the foundation for the ultimate increase of validamycin production.
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  • 刊出日期:  2018-06-24

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