tyrB-2 and phhC genes of Pseudomonas putida encode aromatic amino acid aminotransferase isozymes: evidence at the protein level.

Two Pseudomonas putida aminotransferases (ArAT I and ArAT II) that exhibit activity toward L-tryptophan were purified 104- and 395-fold using a six-stage purification procedure involving ammonium sulfate fractionation and chromatographic separation on phenyl-Sepharose, Sephadex G-100 superfine, DEAE-cellulose and Protein-Pack Q8 HR columns. Mass spectrometry analysis resulted in the identification of 27 and 20 % of the total ArAT I and ArAT II amino acid sequences. In addition, N-terminal sequence fragments of ArAT I and ArAT II were determined using the Edman degradation method. Based on the analyses performed, the studied proteins were identified as products of the tyrB-2 and phhC genes, and the presence of these genes in the investigated bacterial strain was confirmed using molecular biology methods. Extensive analysis of the substrate specificities of ArAT I and ArAT II revealed that both enzymes most efficiently catalyzed reactions involving aromatic amino acids and 2-oxoacids followed by dicarboxylic compounds. The best substrates for ArAT I and ArAT II were L-phenylalanine and phenylpyruvate. Based on these results, the studied proteins were classified as aromatic amino acid aminotransferase isozymes.