We discovered a d-phenylserine deaminase that catalyzed the pyridoxal 5'-phosphate (PLP)-dependent deamination reaction from d-threo-phenylserine to phenylpyruvate in newly isolated Arthrobacter sp. TKS1. The enzyme was partially purified, and its N-terminal amino acid sequence was analyzed. Based on the sequence information, the gene encoding the enzyme was identified and expressed in Escherichia coli. The expressed protein was purified to homogeneity and characterized. The enzyme consisted of two identical 46-kDa subunits and showed maximum activity at pH 8.5 and 55A degrees C. The enzyme was stable in the range of pH 7.5 to pH 8.5 and up to 50A degrees C. The enzyme acted on the d-forms of beta-hydroxy-alpha-amino acids, such as d-threo-phenylserine (K (m), 19 mM), d-serine (K (m), 5.8 mM), and d-threonine (K (m), 102 mM). As l-threonine, d-allo-threonine, l-allo-threonine, and dl-erythro-phenylserine were inert, the enzyme could distinguish d-threo-form from among the four stereoisomers of phenylserine or threonine. The enzyme was activated by ZnSO4, CuSO4, BaCl2, and CoCl2 and strongly inhibited by phenylhydrazine, sodium borohydride, hydroxylamine, and dl-penicillamine. The enzyme exhibited absorption maxima at 280 and around 415 nm. The enzyme has an N-terminal domain similar to that of alanine racemase, which belongs to the fold type III group of pyridoxal enzymes.