An inducible phenylserine aldolase (L-threo-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of L-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of Pseudomonas putida 24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5 phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on L-threo-3-phenylserine (K m , 1.3 mM), L-erythro-3-phenylserine (K m , 4.6 mM), L-threonine (K m , 29 mM), and L-allo-threonine (K m , 22 mM). In the reverse reaction, threo-and erythro-forms of L-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from Pseudomonas putida 24-1 was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5-phosphate.Much attention has been paid to 3-hydroxy-2-amino acids as components of antibiotics and immunosuppressants (5-7, 9, 18, 19, 32-34) and as a drug for Parkinson's disease therapy (21), and enzymatic syntheses of 3-hydroxy-2-amino acids with L-and D-threonine aldolases have been done extensively (5,6,9,18,19,(32)(33)(34).Phenylserine aldolase (L-threo-3-phenylserine benzaldehydelyase, EC 4.1.2.26) catalyzes the reversible conversion of Lthreo-and L-erythro-3-phenylserine to benzaldehyde and glycine. Although the occurrence of phenylserine aldolase in animals has been reported previously (2), the enzyme has not been purified and characterized. Since serine hydroxymethyltransferase (SHMT) (27,28,30) and threonine aldolase (13)(14)(15)(16) show the phenylserine aldolase activity, it has been thought that the phenylserine aldolase activity is due to SHMT or threonine aldolase. During the course of a study on microbial metabolism of DL-threo-3-phenylserine, we found a phenylserine aldolase activity in a soil bacterium identified as Pseudomonas putida 24-1. To use the enzyme for the synthesis of 3-hydroxy-2-amino acids, we purified the enzyme to homogeneity from the bacterium and obtained evidence that the enzyme is an inducible phenylserine aldolase but not SHMT and threonine aldolase. This paper presents the first identification of phenylserine aldolase from P. putida 24-1 and its enzymologic characteristics as well as cloning and overexpression of its gene in Escherichia coli. MATERIALS AND METHODSMaterials. DL-threo-3-Phenylserine, DL-t...