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Enzyme Research
Volume 2010, Article ID 597010, 10 pages
http://dx.doi.org/10.4061/2010/597010
Research Article

Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

1The United Graduate School of Agricultural Science, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan
2Faculty of Agriculture, Kochi University, B200 Monobe, Nankoku, Kochi 783-8502, Japan
3Corporate Research Center, R&D Management, Daicel Chemical Industries, Ltd., 1239 Shinzaike, Aboshi-ku, Himeji 671-1283, Japan
4Himeji Research Center, Daicel Chemical Industries, Ltd., 1239 Shinzaike, Aboshi-ku, Himeji 671-1283, Japan
5Science Research Center, Kochi University, B200 Monobe, Nankoku, Kochi 783-8502, Japan

Received 29 July 2009; Revised 28 October 2009; Accepted 29 November 2009

Academic Editor: Paul Engel

Copyright © 2010 Sakuko Ueshima et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The gene encoding D-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded L-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed L-phenylserine dehydrogenase activity. The enzymological properties and primary structure of L-phenylserine dehydrogenase (ORF3) were quite different from those of D-phenylserine dehydrogenase previously reported. L-Phenylserine dehydrogenase catalyzed the -dependent oxidation of the -hydroxyl group of L- -phenylserine. L-Phenylserine and L-threo-(2-thienyl)serine were good substrates for L-phenylserine dehydrogenase. The genes encoding L-phenylserine dehydrogenase and D-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and .