N-methyl-L-amino acid dehydrogenase from Pseudomonas putida. A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily.
We found N-methyl-L-amino acid dehydrogenase activity in various bacterial strains, such as Pseudomonas putida and Bacillus alvei, and cloned the gene from P. putida ATCC12633 into Escherichia coli. The enzyme purified to homogeneity from recombinant E. coli catalyzed the NADPH-dependent formation of N-alkyl-L-amino acids from the corresponding alpha-oxo acids (e.g. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (e.g. methylamine, ethylamine, and propylamine). Ammonia was inert as a substrate, and the enzyme was clearly distinct from conventional NAD(P)-dependent amino acid dehydrogenases, such as alanine dehydrogenase (EC 1.4.1.1). NADPH was more than 300 times more efficient than NADH as a hydrogen donor in the enzymatic reductive amination. Primary structure analysis revealed that the enzyme belongs to a new NAD(P)-dependent oxidoreductase superfamily, the members of which show no sequence homology to conventional NAD(P)-dependent amino acid dehydrogenases and opine dehydrogenases.
Mihara
Hisaaki
H
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Muramatsu
Hisashi
H
Kakutani
Ryo
R
Yasuda
Mari
M
Ueda
Makoto
M
Kurihara
Tatsuo
T
Esaki
Nobuyoshi
N
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D009249:NADP; D009252:NADPH Dehydrogenase