中国药科大学学报2018，Vol.49Issue(3)：360-368,9.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
Characterization of glutamate dehydrogenase SHJG 7666 from Streptomyces hygroscopicus 5008
摘要
Abstract
Glutamate dehydrogenase (GDH)a key enzyme in the nitrogen metabolism pathway catalyzes the con-version between α-ketoglutarate and glutamate reversibly using NAD(P)H as a cofactor. Based on genomic stud-ies,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 vali-damycin. 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 dinu-cleotide binding motif. Further homologous modeling and structural comparison revealed that SHJG_7666 con-tained conserved Lys60,Lys78and Asp120catalytic functional sites and ligand binding residues Ser36,Gly38,Gln119 and 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 Kmvalue of (25. 3 ±9. 1)μmol/L and kcatof (3 ±0. 8)×10 -5s-1for 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 vali-damycin 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Key words
glutamate dehydrogenase/nitrogen metabolism/validamycin/homologous modeling/characterization of enzyme分类
生物科学引用本文复制引用
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):360-368,9.基金项目
国家自然科学基金资助项目(No. 31620103901,No. 31770098)This study was supported by the National Natural Science Foundation of China （No. 31620103901,No. 31770098）
