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 Lys
60, Lys
78 and Asp
120 catalytic functional sites and ligand binding residues Ser
36, Gly
38, Gln
119and Asp
166, Asn
300, Ala
330. 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.