Characterization of glutamate dehydrogenase SHJG_7666 from<i> Streptomyces hygroscopicus</i> 5008

LIU Zhangmin; CHAO Jiajia; FENG Yan; LI Qian; CUI Li

doi:10.11665/j.issn.1000-5048.20180316

Journal of China Pharmaceutical University > 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

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Characterization of glutamate dehydrogenase SHJG_7666 from Streptomyces hygroscopicus 5008

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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 K_m 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.
- glutamate dehydrogenase,
- nitrogen metabolism,
- validamycin,
- homologous modeling,
- characterization of enzyme

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