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

Abstract EN

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 NAD+-dependent oxidation of the β-hydroxyl group of l-β-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 CO2.