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.

Biosynthesis of the β-Methylarginine Residue of Peptidyl Nucleoside Arginomycin in Streptomyces arginensis NRRL 15941

“…The inconsistency between the in vivo catalysis and the in vitro catalysis by ArgM and ArgN raised two possibilities: (i) another aminotransferase in E. coli participated in the transamination from glutamate to 5-guanidino-3-methyl-2-oxopentanoic acid and (ii) the amino group donor is other than glutamate in the second transamination for ArgM. We first introduced a transaminase from E. coli BL21(DE3), named TyrB, which often converts tyrosine to 4-aminophenylpyruvate in the primary metabolism (17)(18)(19). tyrB from BL21(DE3) was amplified, cloned, and expressed, and the purified TyrB was coupled with ArgM and ArgN to measure their activities toward L-arginine.…”

Whole genome analysis for plant growth promotion profiling of Pantoea agglomerans CPHN2, a non-rhizobial nodule endophyte

“…Among all these Trp-dependent pathways, IAM and IPA pathways are widespread among bacteria (Mano and Nemoto, 2012 ). The IAM pathway is common among phytopathogens such as Pseudomonas syringae, Agrobacterium tumefaciens , and Agrobacterium rhizogenes (Szkop and Bielawski, 2013 ), while most PGPRs such as Pseudomonas putida, Enterobacter cloacae , and Azospirillum brasilense use the IPA pathway for the biosynthesis of IAA. The IPA pathway has been well-characterized in bacteria, and the conversion of indole-3-pyruvate to indole-3-acetaldehyde by ipd C is the rate-limiting step (Zhao, 2010 ).…”