Identification of the Initial Steps in d -Lysine Catabolism in Pseudomonas putida

Abstract

Pseudomonas putida uses L-lysine as the sole carbon and nitrogen source which preferentially requires its metabolism through two parallel pathways. In one of the pathways ␦-aminovalerate is the key metabolite, whereas in the other L-lysine is racemized to D-lysine, and L-pipecolate and ␣-aminoadipate are the key metabolites. All the genes and enzymes involved in the D-lysine pathway, except for those involved in the conversion of D-lysine into ⌬ 1 -piperideine-2-carboxylate, have been identified previously (30). In this study we report that the conversion of D-lysine into ⌬ 1 -piperideine-2-carboxylate can be mediated by a D-lysine aminotransferase (PP3590) and a D-lysine dehydrogenase (PP3596). From a physiological point of view PP3596 plays a major role in the catabolism of D-lysine since its inactivation leads to a marked reduction in the growth rate with L-or D-lysine as the sole carbon and nitrogen source, whereas inactivation of PP3590 leads only to slowed growth. The gene encoding PP3590, called here amaC, forms an operon with dpkA, the gene encoding the enzyme involved in conversion of ⌬ 1 -piperideine-2-carboxylate to L-pipecolate in the D-lysine catabolic pathway. The gene encoding PP3596, called here amaD, is the fifth gene in an operon made up of seven open reading frames (ORFs) encoding PP3592 through PP3597. The dpkA amaC operon was transcribed divergently from the operon ORF3592 to ORF3597. Both promoters were mapped by primer extension analysis, which showed that the divergent ؊35 hexamers of these operon promoters were adjacent to each other. Transcription of both operons was induced in response to L-or D-lysine in the culture medium.Pseudomonas putida KT2440, a derivative of P. putida mt-2 from which the TOL plasmid was cured (15, 25), can grow on proline, lysine, glutamate, and other amino acids as the sole carbon and nitrogen source. The ability to assimilate these compounds confers a selective advantage by enabling the strain to grow in the rhizosphere of a number of plants, where these amino acids form part of the root exudates (4, 13, 21, 23).Pioneering work on bacterial lysine metabolism (17) helped establish two parallel catabolic pathways (Fig. 1), starting with the interconversion by a racemase of both enantiomers of lysine. L-Lysine catabolism proceeds through the so-called aminovalerate (AMV) pathway and involves the following steps: L-lysine 3 ␦-aminovaleramide 3 ␦-AMV 3 glutarate semialdehyde 3 glutarate, which is then channeled into the Krebs cycle via glutaryl-and acetyl-coenzyme A (CoA). On the other hand, D-lysine catabolism proceeds via a series of cyclized intermediates which are necessary to regenerate an ␣-amino acid and comprise the following metabolites (aminoadipate [AMA] pathway): D-lysine 3 ␣-keto-ε-amino caproate 3 ⌬ 1 -piperideine-2-carboxylate 3 pipecolate 3 ⌬ 1 -piperideine-6-carboxylate 3 ␣-amino-␦-formylcaproate 3 ␣-AMA 3 ␣-ketoadipate. The latter is transformed to ␣-ketoglutarate via a series of CoA derivatives. These steps were established at the biochemical le...

Amino Acid Racemization in Pseudomonas putida KT2440

“…Other studies have described the catabolic versatility of P. putida KT2440, yet a comprehensive analysis of growth on D-and L-amino acids as the sole carbon and nitrogen source has not been reported (12,31,32). To assess the potential for racemization of D-amino acids, we performed a series of growth studies on P. putida KT2440 in which D-and L-amino acids were provided as the sole carbon and nitrogen source (Fig.…”

“…They may also be considered models in D-amino acid biology. Recent work in Pseudomonas aeruginosa PAO1 and Pseudomonas putida KT2440 has revealed unique mechanisms by which D-amino acids are catabolized (12,13,22). Here we build on this work by assessing the D-and L-amino acid catabolic capacity of P. putida KT2440, employing a functional screen to identify genes involved in D-amino acid metabolism, characterizing the enzymes involved in amino acid racemization, and identifying key differences in the genomics of D-amino acid metabolism between related pseudomonads.…”

“…In this case, the bacterium is shown to synthesize the specific D-amino acid of note, but little is known about how this is accomplished. Further, bacterial catabolism of certain D-amino acids is noted and may be important for colonization of D-amino-acidrich environments (12,13).…”

l -Lysine Catabolism Is Controlled by l -Arginine and ArgR in Pseudomonas aeruginosa PAO1

“…Transamination of L-lysine by AruH or D-lysine by DauA leads to ␣-keto-ε-aminohexanoate, which is spontaneously converted into its cyclic form ⌬1-piperideine-2-carboxylate at a physiological pH (12). Recent studies of P. putida suggest further degradation of this molecule into L-pipecolate by the reductase DpkA (PP3591) (27). However, viability of this pathway is still unde- on May 11, 2018 by guest http://jb.asm.org/ fined in P. aeruginosa since the dpkA orthologue is not present on the chromosome, the corresponding biochemical reaction cannot be detected, and no growth is detectable using D-lysine as a carbon source (5).…”

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

“…1) has been proposed to degrade D-Lys to 2-aminoadipate, which is then further degraded to a-ketoglutarate to enter the Krebs cycle. Several genes encoding enzymes of this proposed pathway in P. putida KT2440 have been identified, including PP3590 for D-Lys transaminase, PP3596 for D-Lys dehydrogenase, dpkA for D1-piperideine-2-carboxylate reductase (Muramatsu et al, 2005;Revelles et al, 2005Revelles et al, , 2007, and amaB and amaA in conversion of pipecolate to 2-aminoadipate (Revelles et al, 2005). P. aeruginosa does not possess the lysine racemase; however, L-Lys catabolism can potentially be connected to the D-Lys pathway via an arginine-pyruvate transaminase AruH (Chou et al, 2010;Yang & Lu, 2007a).…”