title: Amino acid racemization in Pseudomonas putida KT2440.
abstract: D-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k(cat)/K(m) values with l- and d-lysine were 3 orders of magnitude greater than the k(cat)/K(m) values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher k(cat)/K(m) values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.
authors.affiliation: Department of Plant and Soil Sciences, College of Agriculture, University of Kentucky, Lexington, Kentucky, USA.
authors.forename: Atanas D
authors.initials: AD
authors.lastname: Radkov
authors.forename: Luke A
authors.initials: LA
authors.lastname: Moe
mesh_terms: D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism; D019295:Computational Biology; D016680:Genome, Bacterial; D007536:Isomerism; D007700:Kinetics; D009584:Nitrogen / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D002244:Carbon; D000591:Amino Acid Isomerases; D009584:Nitrogen