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.

Molecular characterization of lysR-lysXE, gcdR-gcdHG and amaR-amaAB operons for lysine export and catabolism: a comprehensive lysine catabolic network in Pseudomonas aeruginosa PAO1

“…The presence of a lysine racemase has been reported in P. putida to convert L-Lys to D-Lys (Radkov & Moe, 2013;Revelles et al, 2005). The catabolic pathway for D-Lys catabolism in this organism ( Fig.…”

Elucidation of the d‐lysine biosynthetic pathway in the hyperthermophile Thermotoga maritima

“…d ‐Ser is incorporated into peptidoglycan in vancomycin‐resistant bacteria , but in most bacteria it is toxic because it inhibits protein synthesis , induces the SOS response, and down‐regulates the type III secretion system . In those species, d ‐Ser is decomposed to pyruvate by d ‐Ser dehydratase (DsdA; http://www.chem.qmul.ac.uk/iubmb/enzyme/EC4/3/1/18.html) and MetC , or converted to the l ‐enantiomer by amino acid racemase . In addition, TM1597 was able to catalyze the epimerization of ll ‐ and meso ‐Dpm, possibly due to the similar structures of Lys and Dpm.…”

Building peptidoglycan inside eukaryotic cells: A view from symbiotic and pathogenic bacteria

“…By contrast, most of the cytosolic reactions required for the synthesis and flip of the lipid II precursor are catalyzed by unique essential enzymes. The exceptions are l ‐Ala and L‐Glu racemases, which are encoded by more than one gene in most Gram‐negative and Gram‐positive bacteria (Kang et al, ; Oh, Richter, Missiakas, & Schneewind, ; Radkov & Moe, ).…”