tailieunhanh - Báo cáo khoa học: N-Methyl-L-amino acid dehydrogenase from Pseudomonas putida A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily

We foundN-methyl-l-amino acid dehydrogenase activity in various bacter-ial strains, such as Pseudomonas putidaandBacillus alvei, and cloned the gene fromP. putidaATCC12633 intoEscherichia coli. The enzyme purified to homogeneity from recombinant E. colicatalyzed the NADPH-dependent formation of N-alkyl-l-amino acids from the corresponding a-oxo acids (. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (. methylamine, ethylamine, and propylamine). | ềFEBS Journal N-Methyl-L-amino acid dehydrogenase from Pseudomonas putida A novel member of an unusual NAD P -dependent oxidoreductase superfamily II I nn i 1x I I I It ÍI 1. n I I 1nfl xs I 2 n fl I . II I 2 Hisaaki Mihara Hisashi Muramatsu Ryo Kakutani Mari Yasuda Makoto Ueda Tatsuo Kurihara1 and Nobuyoshi Esaki1 1 Institute for ChemicalResearch Kyoto University Uji Kyoto 611-0011 Japan 2 Yokohama Research Center Mitsubishi ChemicalCorp. Yokohama Japan Keywords methylamine NADPH N-methyl-L-amino acid dehydrogenase N-methyl-L-amino acid Pseudomonas putida 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 Correspondence N Esaki Institute for ChemicalResearch Kyoto University Uji Kyoto 611-0011 Japan Fax 81 774 38 3248 Tel 81 774 38 3240 E-mail esaki@ formation of N-alkyl-L-amino acids from the corresponding a-oxo acids . pyruvate phenylpyruvate and hydroxypyruvate and alkylamines . 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 . NADPH was more than 300 times more efficient than NADH as Note Equivalent first authors. 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 Database Nucleotide sequence data reported are available in the DDBJ EMBL GenBank databases under the accession number AB190215. homology to conventional NAD P -dependent amino acid dehydrogenases and opine dehydrogenases. Received 8 October 2004 revised 12 December 2004 accepted 22 December 2004 doi