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.

Fig 1

Ability of Pseudomonas putida KT2440 to use d- and l-amino acids as the sole source of carbon and nitrogen. The bar of each color represents the net increase in growth during the designated time period. The absence of a bar of a particular color signifies a lack of growth during the corresponding time period. Cultures were grown in PG liquid minimal medium. Average values are the result of three replicates.

Fig 2

Phylogenetic analysis. (A) Tree of the 50 nonredundant protein sequences most identical to alanine racemase/Alr. (B) Tree of the annotated DadX and Alr from the sequenced genomes in the Pseudomonas Genome Database. Distance bars represent the expected proportion of substitutions per amino acid site. (C) Genomic synteny analysis on regions bordering the alr gene from Pseudomonas putida KT2440 (from the top, listed descending: P. aeruginosa PAO1, P. protegens Pf-5, P. putida KT2440, and P. putida GB-1). Gene annotations are based on the Pseudomonas Genome Database. The H bracket shows the actual DNA length. The gradient scale provides the percent identity for the genomic loci.