Novel phenylalanine dehydrogenases from Sporosarcina ureae and Bacillus sphaericus. Purification and characterization.

NAD+-dependent phenylalanine dehydrogenases were purified 1,500-and 1,600-fold, and crystallized from Sporosarcina ureae SCRC-R04 and Bacillus sphaericus SCRC-R79a, respectively.The purified enzymes were homogeneous as judged by disc gel electrophoresis.The enzyme from S. ureae has a molecular weight of 305,000, while that of B. sphaericus has a molecular weight of 340,000.Each is probably composed of eight subunits identical in molecular weight.The S. ureae enzyme showed a high substrate specificity in the oxidative deamination reaction acting on L- phenylalanine, while that of B. sphaericus acted on Lphenylalanine and L-tyrosine.The enzymes had lower substrate specificities in the reductive amination reaction acting on a-keto acids.The Sporosarcina enzyme acted on phenylpyruvate, a-ketocaproate, a- keto-y-methylthiobutyrate and p-hydroxyphenylpyruvate.The Bacillus enzyme acted on p-hydroxyphenylpyruvate, phenylpyruvate, and a-keto-y-methylthiobutyrate.The enzyme from B. sphaericus catalyzes the transfer of pro-S (B) hydrogen from NADH.NAD+-dependent phenylalanine dehydrogenase was first found to occur in Breuibacterium sp. by Hummel et al. in 1984(1).The enzyme catalyzes reversible oxidative deamination acting on L-phenylalanine.Much attention is being paid to the enzyme not only because its occurrence was completely unknown until the discovery by Hummel et al., but because it appears to be useful as an industrial catalyst in the asymmetric synthesis of L-phenylalanine and related L-amino acids from their keto analogs (2).Enzymes that catalyze this type of reaction include glutamate dehydrogenase (EC 1.4.1.2-4),alanine dehydrogenase (EC 1.4.1.1),and leucine dehydrogenase (EC 1.4.1.9),etc. Glutamate dehydrogenase is widely distributed and few organisms lack this enzyme.Glutamate dehydrogenase of animal origin functions with both NAD' and NADP+, whereas most of the other organisms possess the enzymes specific either for NAD+ or NADP+ (3).The distribution of alanine dehydrogenase (4-9) and leucine dehydrogenase (9, 10) is limited to microorganisms.Both of them are NAD+-dependent, and high activities are observed in Bacillus species, namely in B. sphaericus I F 0 3525 (4, 10).In a study of the stereoselectivity in hydride transfer from the C-4 of nicotinamide ring, Alizade et al. (11) and Ohshima and Soda (4) showed that Bacillus alanine dehydrogenase is A-stereospecific in the hydride transfer.However, other