Valine dehydrogenase from Streptomyces albus: gene cloning, heterologous expression and identification of active site by site-directed mutagenesis.
A gene encoding valine dehydrogenase (Vdh) has been cloned from Streptomyces albus, a salinomycin producer, and expressed in Escherichia coli. The S. albus Vdh is composed of 364 amino acids that showed high homology with several other amino acid dehydrogenases as well as Vdhs from Streptomyces spp. and leucine and phenylalanine dehydrogenases (Ldh and Pdh) from Bacillus spp. A protein of 38 kDa, corresponding to the approximate mass of the predicted S. albus Vdh product (38.4 kDa) exhibiting specific Vdh activity, was observed when the S. albus vdh gene was overexpressed in E. coli under the controlled T7 promoter and was subsequently purified to homogeneity. Among branched- and straight-chain amino acids, L-valine and L-alpha-aminobutyrate were the preferred substrates for the enzyme. Lys-79 and Lys-91 of S. albus Vdh were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydrogenase sequences. To elucidate the functional roles of the lysyl residues, the Lys residues have individually been replaced with Ala by site-directed mutagenesis. Kinetic analyses of the Lys-79 and Lys-91-mutated enzymes revealed that they are involved in the substrate binding site and catalysis, respectively, analogous to the corresponding residues in the homologous Ldh and Pdh.
Hyun
C G
CG
Department of Biological Science, Institute of Bioscience and Biotechnology, Myong Ji University, Yongin, South Korea.
Kim
S S
SS
Park
K H
KH
Suh
J W
JW
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014633:Valine / Q000378:metabolism*; D050802:Valine Dehydrogenase (NADP+)
D000594:Amino Acid Oxidoreductases; D050802:Valine Dehydrogenase (NADP+); D014633:Valine