title: tyrB-2 and phhC genes of Pseudomonas putida encode aromatic amino acid aminotransferase isozymes: evidence at the protein level.
abstract: 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.
authors.affiliation: Department of Biochemistry, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Poland. michal_szkop@sggw.pl
authors.forename: Michał
authors.initials: M
authors.lastname: Szkop
authors.forename: Wiesław
authors.initials: W
authors.lastname: Bielawski
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000378:metabolism; D010667:Phenylpyruvic Acids / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D010667:Phenylpyruvic Acids; D010649:Phenylalanine; D000637:Transaminases; C019729:aromatic amino acid aminotransferase; C031606:phenylpyruvic acid