Identification of essential amino acid residues in valine dehydrogenase from Streptomyces albus.
Cys-29 and Cys-251 of Streptomyces albus valine dehydrogenase (ValDH) were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydroganase sequences. To ascertain the functional role of these cysteine residues in S. albus ValDH, site-directed mutagenesis was performed to change each of the two residues to serine. Kinetic analyses of the enzymes mutated at Cys-29 and Cys-251 revealed that these residues are involved in catalysis. We also constructed mutant ValDH by substituting valine for leucine at 305 by site-directed mutagenesis. This residue was chosen, because it has been proposed to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). Kinetic analysis of the V305L mutant enzyme revealed that it is involved in the substrate binding site. However it displayed less activity than the wild type enzyme toward all aliphatic and aromatic amino acids tested.
Division of Bioscience & Bioinformatics, MyongJi University, Yongin 449-728, Republic of Korea.
Chang-Gu
CG
Hyun
Sang-Suk
SS
Kim
Joo-Won
JW
Suh
D000594:Amino Acid Oxidoreductases; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000601:Amino Acids, Essential / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000737:chemistry; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D050802:Valine Dehydrogenase (NADP+) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000601:Amino Acids, Essential; D012694:Serine; D000594:Amino Acid Oxidoreductases; D050802:Valine Dehydrogenase (NADP+); D003545:Cysteine