Characterization of glutamate dehydrogenase SHJG 7666 from Streptomyces hygroscopicus 5008
10.11665/j.issn.1000-5048.20180316
- VernacularTitle:吸水链霉菌5008中谷氨酸脱氢酶SHJG_7666的性质表征
- Author:
Zhangmin LIU
1
;
Jiajia CHAO
;
Yan FENG
;
Qian LI
;
Li CUI
Author Information
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
- Keywords:
glutamate dehydrogenase;
nitrogen metabolism;
validamycin;
homologous modeling;
characterization of enzyme
- From:
Journal of China Pharmaceutical University
2018;49(3):360-368
- CountryChina
- Language:Chinese
-
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.
