The Putative Malate/Lactate Dehydrogenase from Pseudomonas putida Is an NADPH-dependent Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Involved in the Catabolism of d-Lysine and d-Proline

Abstract

A Pseudomonas putida ATCC12633 gene, dpkA, encoding a putative protein annotated as malate/L-lactate dehydrogenase in various sequence data bases was disrupted by homologous recombination. The resultant dpkA ؊ mutant was deprived of the ability to use D-lysine and also D-proline as a sole carbon source. The dpkA gene was cloned and overexpressed in Escherichia coli, and the gene product was characterized. The enzyme showed neither malate dehydrogenase nor lactate dehydrogenase activity but catalyzed the NADPH-dependent reduction of such cyclic imines as ⌬ 1 -piperideine-2-carboxylate and ⌬ 1 -pyrroline-2-carboxylate to form L-pipecolate and L-proline, respectively. NADH also served as a hydrogen donor for both substrates, although the reaction rates were less than 1% of those with NADPH. The reverse reactions were also catalyzed by the enzyme but at much lower rates. Thus, the enzyme has dual metabolic functions, and we named the enzyme ⌬ 1 -piperideine-2-carboxylate/⌬ 1 -pyrroline-2-carboxylate reductase, the first member of a novel subclass in a large family of NAD(P)-dependent oxidoreductases.Lactate dehydrogenase (LDH) 1 and malate dehydrogenase (MDH) comprise a complex protein superfamily with multiple enzyme homologs found in eubacteria, Archaea, and eukaryotes (1). They catalyze NAD(P)-dependent interconversions between lactate and pyruvate and between malate and oxaloacetate, respectively. However, a new class of NAD(P)-dependent malate/L-lactate dehydrogenases with no sequence homology to "orthodox" MDH or LDH has been demonstrated (2, 3). Moreover, they have no Rossmann fold, which is a sixstranded parallel ␤-sheet core surrounded on both sides by helices (4), in their NAD(P)-binding domains in contrast to the orthodox proteins. Consequently, the new class of malate/Llactate dehydrogenase family has been distinguished from the orthodox one as shown in protein data bases such as InterPro (www.ebi.ac.uk/interpro/index.html) (5) and Pfam (www.sanger. ac.uk/Software/Pfam/) (6). However, many of the family members are annotated without functional evidence as MDH or LDH only because of their sequence similarities to those of a few enzymes such as MDH from Methanothermus fervidus (2) and LDH from Alcaligenes eutrophus (3). In fact, three hypothetical MDHs of this family have been shown to be (S)-2-hydroxyacid dehydrogenase (7), ureidoglycolate dehydrogenase (8), and 2,3-diketo-L-gulonate reductase (9). The NAD(P) dependence is common to them, but no other functional similarities can be assigned among them. Thus, we expect the occurrence of various other proteins with new functions in this family even though they are annotated as MDH (or LDH) in protein data bases.Pseudomonas strains use both enantiomers of lysine as a sole source of carbon (as well as nitrogen) (10, 11). L-Lysine is catabolized by Pseudomonas putida through the ␦-aminovalerate pathway (11), whereas D-lysine is catabolized through the pipecolate pathway involving a series of reactions through six-carbon cyclic intermediates (12, 13) (Fig. 1)...

Molecular characterization of LhpR in control of hydroxyproline catabolism and transport in Pseudomonas aeruginosa PAO1

“…On the contrary, the dpkA mutant lost the ability to grow on D-Pro as the sole nitrogen source, and this growth defect was complemented by the dkpA clone. This result was consistent with the reported functions of dkpA in P. putida (Muramatsu et al, 2005).…”

“…The PA1255 gene following lhpPHMNO codes for a putative amino acid racemase, and was designated as lhpK here in this study. The peptide encoded by the stand-alone PA1252 gene exhibited moderate sequence similarity to DpkA of P. putida KT2440 that has been reported as an NADP-dependent D1-piperideine-2-carboxylate/D1-pyrroline-2-carboxylate reductase in the catabolism of D-lysine and D-proline (Muramatsu et al, 2005). Also listed in the table were PA1261 (lhpR) and PA1269 encoding two transcriptional regulators, although they were not induced by L-Hyp or D-Hyp.…”

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

“…One missing link in the converged pathway is the enzyme converting D9-piperidine-2-carboxylate to L-Pip. One potential candidate for this enzyme was encoded by PA1252, based on its sequence similarity to DpkA (PP3591) of P. putida KT2440 that has been previously shown to catalyse this reaction in D-Lys and D-Pro catabolism (Muramatsu et al, 2005;Revelles et al, 2005). From the results of transcriptome analysis (Table 3), the PA1252 gene was found not to be inducible by L-Lys, D-Lys or L-Pip.…”

“…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).…”

Crystal Structures of Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Belonging to a New Family of NAD(P)H-dependent Oxidoreductases

“…These two enzymes and their homologs have thus been annotated as new MDH/LDHs different from the conventional MDH/LDH. Recent biochemical and genetic studies have revealed that some of the gene products homologous to proteins belonging to the new MDH/LDH family catalyze reactions other than those involved in MDH or LDH activity, as exemplified by L-sulfolactate dehydrogenase (SLDH) (5), ureidoglycolate dehydrogenase (UGDH) (6), 2,3-diketo-L-gulonate reductase (YiaK) (7), and ⌬ 1 -piperideine-2-carboxylate (Pip2C)/ ⌬ 1 -pyrroline-2-carboxylate (Pyr2C) reductase (8). ThenewMDH/LDHfamilyhasnowbeenextendedtothenewNAD(P)Hdependent oxidoreductase family which contains proteins homologous to, but different in activities from, the new family of MDH/LDH proteins (5,9).…”