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Originally published In Press as doi:10.1074/jbc.M411918200 on November 23, 2004

J. Biol. Chem., Vol. 280, Issue 7, 5329-5335, February 18, 2005
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The Putative Malate/Lactate Dehydrogenase from Pseudomonas putida Is an NADPH-dependent {Delta}1-Piperideine-2-carboxylate/{Delta}1-Pyrroline-2-carboxylate Reductase Involved in the Catabolism of D-Lysine and D-Proline*{boxs}

Hisashi Muramatsu{ddagger}§, Hisaaki Mihara{ddagger}§, Ryo Kakutani{ddagger}, Mari Yasuda¶, Makoto Ueda¶, Tatsuo Kurihara{ddagger}, and Nobuyoshi Esaki{ddagger}||

From the {ddagger}Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan and the Yokohama Research Center, Mitsubishi Chemical Corporation, Yokohama 227-8502, Japan

Received for publication, October 20, 2004 , and in revised form, November 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {Delta}1-piperideine-2-carboxylate and {Delta}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 {Delta}1-piperideine-2-carboxylate/{Delta}1-pyrroline-2-carboxylate reductase, the first member of a novel subclass in a large family of NAD(P)-dependent oxidoreductases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 six-stranded parallel {beta}-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/L-lactate 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 {delta}-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). {Delta}1-Piperideine-2-carboxylate (Pip2C) is reduced to L-pipecolate by Pip2C reductase (EC 1.5.1.21 [EC] ), which has been purified and characterized by Payton and Chang (14). The reduction of Pip2C is also catalyzed by {Delta}1-pyrroline-2-carboxylate (Pyr2C) reductase (EC 1.5.1.1 [EC] ), which inherently acts on Pyr2C, a five-membered ring homolog of Pip2C, to form L-proline in mammals, plants, and microorganisms (15–18). However, it is not yet clear whether such a dual function of Pyr2C reductase is caused by fortuitously broad specificity of the enzyme or of physiological significance.



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  		FIG. 1.
Proposed catabolic pathways for degradation of L- and D-lysine by P. putida. Reactions from D-lysine to {alpha}-ketoadipate represent the pipecolate pathway, and those from L-lysine to glutarate represent the {delta}-aminovalerate pathway (10, 12).

 
We searched for a new enzyme catalyzing the formation of N-methyl-L-phenylalanine from phenylpyruvate and methylamine and found that the enzyme gene is identical to the gene annotated as MDH of the new family of NAD(P)-dependent dehydrogenases described above (19). However, the gene product showed no MDH activity. We have been skeptical about whether N-methyl-L-amino acids are the physiological substrates of the enzyme and carried out gene disruption experiments to clarify this point. Thus, as described here, we have found that the enzyme gene is actually responsible for the catabolism of both D-lysine and D-proline and that the gene product has both Pip2C reductase and Pyr2C reductase activities, which are much higher than the N-methyl-L-amino acid dehydrogenase activity. In addition, multiple sequence alignment analysis has revealed that the enzyme is the first member of a novel subclass in an NAD(P)-dependent oxidoreductase superfamily.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—D-Amino acid oxidase from porcine kidney was purchased from Sigma. Pip2C and Pyr2C were synthesized as described previously (16). Superose 12 and molecular weight marker proteins were obtained from Amersham Biosciences. DEAE-Toyopearl was from Tosoh (Tokyo, Japan). Green-Sepharose was prepared according to a previous method (20). NADH, NADPH, and molecular-weight marker proteins for gel filtration were from Oriental Yeast (Tokyo, Japan). Restriction enzymes and kits for genetic manipulation were from Takara Shuzo (Kyoto, Japan), Toyobo (Osaka, Japan), and New England Biolabs (Beverly, MA). A plasmid, pK18mobsacB, was obtained from the Department of Microbial Genetics, National Institute of Genetics (Japan). All other reagents were of analytical grade from Nacalai Tesque (Kyoto, Japan) and Wako Pure Chemical Industries (Osaka, Japan). DpkA was purified from Escherichia coli BL21(DE3) carrying pDPKA as described previously (19).

Bioinformatic Analysis—Nucleotide and amino acid sequences were obtained from PubMed at NCBI (www.ncbi.nlm.nih.gov/). A homology search was performed with the BLAST program (21) at GenomeNet (www.genome.ad.jp/). Multiple alignments were obtained with the ClustalW program (22) at DDBJ (spiral.genes.nig.ac.jp/homology/). Phylogenetic trees were computed with ClustalW by the neighbor joining method with Kimura's correction (bootstrap scores for 1000 iterations). Trees were displayed using a TreeView program (23).

Targeted Gene Disruption—A P. putida mutant was obtained by disruption of the dpkA gene with the pK18mobsacB plasmid (24). Two gene fragments, DPK1 (0.53 kbp) and DPK2 (0.54 kbp), corresponding to the 5'- and 3'-halves of the dpkA gene were amplified from P. putida ATCC12633 DNA by PCR with primer sets, dpk-f/dpkConA and dpk-r1/dpkConB, respectively (see below). A tetracycline-resistant gene fragment (TcR, 1.35 kbp) was obtained by PCR with a primer set TcBRConA/TcConB and pBR322 as a template. The DPK1, DPK2, and TcR fragments were connected by crossover PCR (25, 26) to yield a 2.4-kbp fragment (DPK1-TcR-DPK2). The resulting fragment was digested with EcoRI and inserted into the EcoRI site of pK18mobsacB to form pK18dpkTc. The resulting plasmid thus contained an insertional disruption of dpkA. pK18dpkTc was introduced into E. coli S17–1 (27) and then into P. putida ATCC12633 by filter mating (28) with selection on an LB medium. Resulting transconjugants were selected for the integration of the tetracycline marker in the chromosome on LB agar containing 50 µg/ml tetracycline and 10% sucrose. The mutant was analyzed by PCR with the dpk-f/dpk-r1 primers to confirm the presence of its disrupted copy of dpkA gene. The primers used are dpk-f, 5'-GGAATTCCATATGTCCGCACCTTCCACCAGCACCG-3'; dpkConA, 5'-CCCATCCACTAAACTTAAACAGAAAGCGATGGGGTTGGTAC-3'; dpk-r1, 5'-GGGAAGCTTTCAGCCAAGCAGCTCTTTCAGG-3'; dpk-ConB, 5'-GGGCAGGGTCGTTAAATAGCGCTGCGCCTTGCGCCGAGCAT-3'; TcBRConA, TGTTTAAGTTTAGTGGATGGGTGACAGCTTATCATCGATAA-3'; and TcConB, 5'-GCTATTTAACGACCCTGCCCTTCTTGGAGTGGTGAATCCG-3'.

Enzyme Assays—The Pip2C/Pyr2C reductase activity of the enzyme was examined in a coupled reaction system containing 100 mM Tris-HCl (pH 8.0), 50 mM D-proline or DL-pipecolate, 18 µM D-amino acid oxidase, 10 mM NADPH, and 7.4 µM of the enzyme at 30 °C for 10 h. The reaction was stopped by addition of trichloroacetic acid (2%), and the supernatant solution obtained after centrifugation was subjected to HPLC analysis: column, CHIRALPAK WE (Daicel, Tokyo, Japan); mobile phase, 2 mM CuSO4; flow rate, 0.5 ml/min; detection, 254 nm; temperature, 50 °C. Alternatively, Pip2C/Pyr2C reductase was assayed with a decrease in absorbance at 340 nm because of the consumption of NADPH at 30 °C in a 1-ml reaction mixture containing 0.062 mM Pip2C (or Pyr2C), 0.15 mM NADPH, and 100 mM Tris-HCl at pH 8.0 for Pip2C or pH 7.0 for Pyr2C. The reverse reaction was followed in the same manner except that 20 mM L-pipecolate (or L-proline), 0.25 mM NADP+, and 100 mM Bis-tris propane (pH 10.0) were substituted for the corresponding components.

Kinetic Analysis—Initial rates of the enzyme reaction were measured with varying concentrations of one substrate and a fixed concentration of the other substrate. Substrate concentration ranges causing substrate inhibition were judged by application of the data to the hyperbolic Michaelis-Menten equation with the Kaleida Graph software (Adelbeck Software, Reading, PA) or the IGOR Pro software (Wave-Metrics, Inc., Lake Oswego, OR). Data obtained in the range without substrate inhibition were fitted to equation 1, which conforms to a sequential mechanism. Ka and Kb represent the Michaelis constants for NADPH (A) and Pip2C (or Pyr2C) (B), respectively. Kia is the dissociation constant for the enzyme-NADPH complex. Product inhibition studies were performed in the presence of various concentrations of NADP+, and the data were fitted to equations 2, 3, 4 corresponding to competitive, uncompetitive, and noncompetitive inhibition patterns, respectively. P is the concentration of the product; Kis is the inhibition constant from the slope term; and Kii is the inhibition constant from the intercept term. The apparent substrate inhibition constant (Ki) for Pyr2C was determined by fitting the data to equation 5.

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(Eq. 5)

Western Blotting—A polyclonal antibody against DpkA was generated in a rabbit. Proteins were separated by SDS-PAGE and blotted onto an Immobilon-P membrane (Millipore, Bedford, MA). DpkA was detected with the anti-DpkA antibody and ECL Western blotting detection reagents (Amersham Biosciences).

Other Analytical Methods—Protein quaternary structure was analyzed by an AKTAexplorer system (Amersham Biosciences) using a YMC-Pack Diol 200 column (YMC Co., Ltd., Kyoto, Japan). The column was equilibrated and operated at a flow rate of 1.0 ml/min with a 0.1 M potassium phosphate buffer (pH 7.0) containing 0.2 M NaCl. The protein standards used were cytochrome c, myokinase, enolase, lactate dehydrogenase, and glutamate dehydrogenase from Oriental Yeast, Osaka, Japan. The N-terminal amino acid sequence of the enzyme was determined with an automated Shimadzu PPSQ10 protein sequencer (Kyoto, Japan). The nucleotide sequence of DNA was determined with an Applied Biosystems 370A DNA sequencer (Foster City, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The dpkA Gene Is Essential for the Catabolism of D-Lysine and D-Proline—We found that a novel enzyme, N-methyl-L-amino acid dehydrogenase, is encoded by a gene named dpkA in P. putida ATCC12633 (19). However, this bacterium was unable to grow on N-methyl-L-alanine as a sole carbon source (Table I), and we speculated that the gene has other physiological functions than the metabolism of N-methyl-L-amino acids. Thus, we carried out gene disruption experiments by introducing a tetracycline-resistant gene (tet) at the center of dpkA (Fig. 2A). A tetracycline-resistant strain obtained had an insertion of 1.35 kbp in dpkA as expected and lacked the ability to produce the DpkA protein (Fig. 2, B and C). The dpkA– strain (termed HM3591) was distinct from the wild-type strain in that neither D-lysine nor D-proline supported the growth as a sole carbon source under the same conditions. Our other finding was that the mutant strain grew only slowly in the medium containing L-lysine as a sole carbon source in contrast to the wild-type strain. No difference was evident between the two strains in other media containing as a sole carbon source L-pipecolate, L-proline, and various D-amino acids except D-lysine and D-proline: D-alanine, D-valine, D-leucine, D-isoleucine, D-serine, D-threonine, D-aspartate, D-glutamate, D-glutamine, D-arginine, and D-phenylalanine (Table I). The retarded growth of the mutant on L-lysine cannot be explained clearly at present, but it is clear that the dpkA gene is essential for the metabolism of D-lysine and D-proline in P. putida.


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  		TABLE I
Growth phenotype of a P. putida dpkA- strain, HM3591

The cells were grown at 30°C in a medium containing 13.6 g/liter KH2PO4, 1.0 g/liter NH4Cl, 1.0 g/liter (NH4)2SO4, 20 mg/liter MgSO4, 1.56 mg/liter Fe(NH4)2(SO4)2·6H2O and 5g/liter glucose. Glucose was omitted or replaced by methylamine·HCl or amino acid indicated. The growth of the cells was monitored with a Bio-Photorecorder TN-1506 (Advantec, Tokyo, Japan).

 



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  		FIG. 2.
Targeted disruption of the dpkA gene by homologous recombination. A, construction of a dpkA-disruptant of P. putida. A disruption plasmid, pK18dpkTc, contained a disrupted derivative of dpkA in which a tetracycline-resistant cassette (tet) was inserted at the center of dpkA. Introduction of pK18dpkTc into the parent strain P. putida ATCC12633 resulted in the disruption of the chromosomal dpkA by homologous recombination. The positions of primer-annealing sites for PCR are indicated by the arrows. B, PCR analysis of the DNA region containing the disrupted dpkA in a mutant strain, HM3591. The sizes of the PCR fragments obtained with the primers shown in A are 1.0 kbp for the parent strain (lane 2) and 2.4 kbp for the dpkA– (HM3591) strain (lane 3). DNA size markers are shown in lane 1. C, Western blot analysis of the crude extracts from the parent (lane 2) and the dpkA– strains (lane 3). The purified enzyme (11 ng) was loaded in lane 1. The DpkA protein was detected with the rabbit polyclonal antiserum raised against the purified protein.

 
DpkA Exhibits Both Pip2C Reductase and Pyr2C Reductase Activities—D-Lysine is catabolized in P. putida through the pipecolate pathway involving a series of reactions through six-carbon alicyclic intermediates as follows: D-lysine -> {alpha}-keto-{epsilon}-aminohexanoate -> Pip2C -> L-pipecolate -> {Delta}1-piperideine-6-carboxylate -> L-{alpha}-aminoadipate (Fig. 1) (12, 13). However, no enzyme genes of this pathway have been identified in P. putida. The dpkA– strain grew on L-pipecolate but not on D-lysine as described above, and we assumed that the gene encodes Pip2C reductase, which catalyzes the reduction of Pip2C to L-pipecolate. We confirmed that this is exactly the case as follows. When racemic pipecolate was used as a substrate and incubated with DpkA purified as described above in the presence of D-amino acid oxidase, D-pipecolate of the racemate disappeared and the amount of L-pipecolate was doubled (data not shown). This indicates that D-pipecolate is converted to L-pipecolate by DpkA probably through Pip2C; D-pipecolate serves as a good substrate of D-amino acid oxidase to be converted to Pip2C, which is then transformed to L-pipecolate by DpkA. When D-proline was substituted for DL-pipecolate in the reaction system, it was completely converted to L-proline (Fig. 3). These results clearly indicate that DpkA acts not only on Pip2C but also on Pyr2C. In fact, both Pip2C and Pyr2C synthesized as described under "Experimental Procedures" served as substrates of DpkA when NADPH oxidation was followed at 340 nm; Pip2C is better than Pyr2C as a substrate (Table II). Moreover, both L-pipecolate and L-proline underwent oxidation with NADP+ as a coenzyme although at much lower rates than the forward reaction (Table II). Namely, the reaction equilibrium favors the direction toward reduction of Pip2C (and Pyr2C). The results in vivo and in vitro together clearly indicate that DpkA has dual metabolic functions as Pip2C reductase (EC 1.5.1.21 [EC] ) and Pyr2C reductase (EC 1.5.1.1 [EC] ). NADH served as the coenzyme in both reactions but was much poorer than NADPH (Table II). The reductive methylamination of pyruvate proceeds at a rate of about a half of that of the Pyr2C reduction (Table II). Similarly, the oxidative deamination of N-methyl-L-alanine is much slower than that of L-proline.



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  		FIG. 3.
HPLC analysis of the product by the DpkA reaction. The reaction mixture contained 100 mM Tris-HCl (pH 8.0), 50 mM D-proline, 18 µM D-amino acid oxidase, 10 mM NADPH, and 7.4 µM DpkA. The reaction was stopped with trichloroacetic acid immediately (A) or at 600 min (B) after the addition of DpkA.

 


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  		TABLE II
NADP(H)- and NAD(H)-dependent reactions catalyzed by DpkA

 
Effects of pH and Temperature on DpkA—The maximum activity of DpkA toward Pip2C occurred at pH 8.0, which was distinctly higher than that toward Pyr2C (pH 7.0) (Fig. 4, A and B). However, we found no clear difference in the maximum pH values in oxidation between L-pipecolate and L-proline; both were pH 10.0 (Fig. 4, C and D). The enzyme showed the highest activity in the 20 mM Tris-HCl buffer (pH 7.0) at 35 °C but was inactivated at a rate of about a 30% decrease in 30 min under the same conditions. The enzyme kept its full activity, however, at 30 °C for at least 30 min.



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  		FIG. 4.
Effect of pH on the activity of DpkA. The pH dependence of the reduction of Pip2C (A), the reduction of Pyr2C (B), the oxidation of L-pipecolate (C), and the oxidation of L-proline (D) catalyzed by DpkA. The assays were carried out with 100 mM Tris-HCl (pH 6.8–9.0) and 100 mM glycine-NaOH (pH 9.0–11.0).

 
Substrate Specificity—The purified enzyme exhibited neither malate dehydrogenase nor lactate dehydrogenase activity regardless of coenzyme forms (NADH or NADPH) and reaction directions. The enzyme exhibited weak oxidation activity toward trans-4-hydroxy-L-proline and L-thioproline (Table III). However, 2-picolinate, pyrazole-3,5-dicarboxylate, cis-4-hydroxy-D-proline, L-azetidine-2-carboxylate, (S)-2-pyrroline-5-carboxylate, piperazine-2-carboxylate, or (S)-2-pyrrolidone-5-carboxylate did not serve as a substrate.


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  		TABLE III
Substrate specificity of DpkA

 
Inhibition by Pyrrole-2-carboxylate—A substrate analog, pyrrole-2-carboxylate inhibited the enzyme; it showed only 10% of its original activity in the presence of 1.0 mM of this inhibitor. Pyr2C reductase from pig is strongly inhibited by N-formylphenylalanine at a Ki value of 0.29 mM (18). However, DpkA was resistant against 1 mM N-formylphenylalanine.

Initial Rate Studies—DpkA was subject to substrate inhibition by Pip2C and Pyr2C (Fig. 5). Their reactions followed Michaelis-Menten kinetics with substrate concentrations lower than 60 µM for Pip2C and 110 µM for Pyr2C. However, the reaction rates decreased with increasing substrate concentration over these ranges. The theoretical curve calculated with equation 5 fitted well with the experimental data for Pyr2C, and an apparent inhibition constant (Ki) was calculated to be 1.2 mM (Fig. 5). However, the experimental data deviated from the calculated curve for Pip2C.



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  		FIG. 5.
Inhibition of DpkA by the substrates Pip2C (A) and Pyr2C (B). The Pip2C (A) or Pyr2C (B) concentrations were changed with the fixed concentration of NADPH at 150 µM. Each line represents a best fit of the data (by non-linear regression analysis) to equation 5.

 
We studied steady-state kinetics with substrate concentrations causing no substrate inhibition: [Pip2C], 8–60 µM; [Pyr2C], 3.7–55 µM. Initial velocities were measured with varying concentrations of NADPH or cyclic imino acid (namely Pip2C or Pyr2C) at several fixed concentrations of the other. By double-reciprocal plots, we obtained families of linear lines with intersecting patterns characteristic of a sequential mechanism (Fig. 6) and determined the kinetic constants with slopes and intercepts (Tables IV and V). The intersecting points occurred at the left of the 1/V axis, and accordingly a sequential rapid-equilibrium ordered mechanism was excluded.



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  		FIG. 6.
Initial velocity patterns for the Pip2C/Pyr2C reductase reaction. The NADPH concentrations were changed with the following fixed concentrations of Pip2C (A): open triangle, 55 µM; closed circle, 37 µM; open square, 18 µM; closed triangle, 11 µM; open circle, 7.4 µM; and closed square, 3.7 µM; or Pyr2C (C): closed circle, 60 µM; open square, 40 µM; closed triangle, 20 µM; closed circle, 12 µM; and closed square, 8 µM. Slopes and 1/v axis intercepts versus [Pip2C] (B) and [Pyr2C] (D) are shown. The assays were carried out in 100 mM Tris-HCl buffer (pH 8.0 for Pip2C and pH 7.0 for Pyr2C) at 30 °C. Each line represents a best fit of the data to equation 1.

 


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  		TABLE IV
Kinetic parameters of DpkA in the forward direction

 


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  		TABLE V
Kinetic parameters of DpkA in the reverse direction

 
Product Inhibition Studies—We studied the kinetic mechanism of DpkA further with respect to product inhibition by Pyr2C and NADPH in the oxidation of L-proline. NADPH was a competitive inhibitor against NADP+, indicating that they bind with the same enzyme form (supplemental Fig. S1). However, no other couples were competitive with one another (Table VI). Both NADPH and Pyr2C were mixed-type noncompetitive inhibitors against L-proline. Pyr2C also served as a mixed-type noncompetitive inhibitor against NADP+. However, the inhibition pattern changed to uncompetitive when the L-proline concentration was increased to a saturating level. These results indicate that the DpkA reaction proceeds through an ordered Bi-Bi mechanism; NADPH and then Pyr2C bind with the enzyme to form a ternary complex, which releases L-proline and then NADP+ to return to the unbound form (Scheme 1).


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  		TABLE VI
Product inhibition of DpkA by L-proline and NADP+ in the reverse direction

 



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  		SCHEME 1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Relationship with Other Family Members—A phylogenetic analysis of DpkA and several proteins involved in the malate/L-lactate dehydrogenase family revealed that these proteins are grouped into at least eight clusters (Fig. 7). The proteins in each subgroup share more than 40% sequence identities with one another. These subfamilies include (i) the DpkA clade containing P. putida DpkA and its close homologs from Pseudomonas syringae and Pseudomonas aeruginosa; (ii) the YiaK clade, which includes 2,3-diketo-L-gulonate reductase (YiaK) from E. coli (9) and its homologs from Haemophilus influenzae and Pasteurella multocida; (iii) the AllD clade involving ureidoglycolate dehydrogenase (AllD) from E. coli (8) and homologs from Salmonella typhimurium and Bacillus subtilis; (iv) the YlbC clade containing eukaryotic homologs with unknown function from Caenorhabditis elegans and Drosophila; (v) thermophilic archaeal proteins from Pyrococcus abyssi and Pyrococcus horikoshii; (vi) the ComC clade, which involves L-sulfolactate dehydrogenases (ComC) from M. fervidus (2), Methanococcus jannaschii (7), and Methanobacterium thermoautotrophicum (29); (vii) the clade including LDH from A. eutrophus (3); and (viii) the clade including YbiC homologs from E. coli and Bradyrhizobium japonicum (Fig. 7). However, the biochemical functions of most family members remain unknown, except those of LDH from A. eutrophus (3), YiaK from E. coli (9), AllD from E. coli (8), and ComCs from Archaea (2, 7, 29). DpkA is the first enzyme in the DpkA clade whose function has been identified. Whether the members of this clade indeed catalyze the reduction of Pip2C (and also of Pyr2C) is under investigation.



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  		FIG. 7.
Unrooted neighbor joining tree of sequences homologous to that of DpkA. Sequence data were taken from data banks and the tree was created with the ClustalW (22) and TreeView (23) programs. The scale bar represents 0.1 amino acid substitution per site. Bootstrap values (1000 bootstrap replicates) are indicated. The sequence of P. putida DpkA is shown in bold. Enzymes whose catalytic activity has been experimentally shown are indicated by asterisks. Accession numbers for the proteins used are as follows. DpkAs: P. syringae, Q883J8 (TrEMBL); P. putida, AB190215 [GenBank] (GenBankTM); P. aeruginosa, Q9I492 (Swiss-Prot). LDHs: E. coli O6, Q8FJM9 (TrEMBL); A. eutrophus, Q07251 [GenBank] (Swiss-Prot). YbiCs: B. japonicum, Q89R55 (TrEMBL); E. coli, P30178 [GenBank] (Swiss-Prot). AllDs: S. typhimurium, Q8ZM60 (TrEMBL); E. coli, P77555 [GenBank] (Swiss-Prot); B, E69852 [GenBank] (PIR). Thermophilic archaeal proteins: P. abyssi, Q9V0D5 (Swiss-Prot); P. horikoshii, O59028 [GenBank] (Swiss-Prot). ComCs: M. fervidus, P16142 [GenBank] (Swiss-Prot); M. thermoautotrophicum, O26290 [GenBank] (Swiss-Prot); M. jannaschii, Q60176 [GenBank] (Swiss-Prot). YlbCs: Drosophila melanogaster, Q8IPT9 (TrEMBL); C. elegans, T20396 [GenBank] (PIR). YiaKs: H. influenzae, P44995 [GenBank] (Swiss-Prot); P. multocida, Q9CLH5 (Swiss-Prot); E. coli, P37672 [GenBank] (Swiss-Prot).

 
Preference for NADPH as the Coenzyme—Recently, the crystal structures of YiaK from E. coli (30) and sulfolactate dehydrogenase from M. jannaschii (SLDH) (31) were determined. The structures of the protein complexes with NAD+ were also analyzed, and the residues directly interacting with NAD+ were identified (30, 31). His44, Asp173, Lys225, and Glu307 of SLDH interacting with NAD+ are completely conserved in YiaK and mostly in DpkA, except Glu307, which is replaced by Arg313 (or Arg314) in DpkA. The side chain of Glu307 in SLDH, which is conserved as Glu306 in YiaK, interacts with the diol group of the ADP-ribose of NADH. The Vmax/Km value for SLDH with NADPH as a coenzyme is less than 2% of that with NADH. Similarly, NADH is much better as a coenzyme of YiaK than NADPH. By contrast, DpkA prefers NADPH to NADH and is the first enzyme with this coenzyme preference among all family members. However, both Arg313 and Arg314 of DpkA are also conserved in other members of the same clade, suggesting that all of them show the same coenzyme specificity as DpkA. It is reasonable to speculate that the positive charge of Arg313 (or Arg314) in DpkA plays an important role for the interaction with the 2'-phosphate group of NADPH.

Physiological Function of DpkA—We found lysine racemase activity in the periplasm of P. putida ATCCC12633 (data not shown). This is consistent with previous findings (32) and suggests a linkage between the catabolic pathways of L-lysine and D-lysine through the racemization of lysine (Fig. 1). However, the growth defect observed with the dpkA-deficient mutant HM3591 on D-lysine as a sole carbon source suggests that DpkA is responsible for the reduction of Pip2C to L-pipecolate in vivo and that D-lysine is catabolized only through the pipecolate pathway. Chang and Adams (10) reported that a mutant of P. putida ATCC 25991 defective of the enzyme converting {delta}-aminovalerate to glutarate semialdehyde (Fig. 1) is unable to grow on L-lysine as a sole carbon source but capable of growing normally on D-lysine. Therefore, even though lysine racemase occurs in P. putida, the catabolic pathways of L-lysine (through the {delta}-aminovalerate pathway) and D-lysine (through the pipecolate pathway) are apparently not linked by the racemase. Such disconnection can also be seen between L-proline and D-proline in P. putida although it has proline racemase encoded by the putative proline racemase gene PP1258 (data not shown). As described above, we found that the mutant HM3591 failed to utilize D-proline as a sole carbon source in contrast to L-proline. Pyr2C is known to be a strong inhibitor of proline racemase (33) and is produced from D-proline by the action of D-amino acid dehydrogenase. However, DpkA is deficient in the mutant, and Pyr2C is probably accumulated in the cells and prevents proline racemase from producing L-proline. Little is known about lysine racemase, in particular, whether Pip2C inhibits it in the same manner as Pyr2C inhibits proline racemase. If this is the case, however, then the above observation with the mutants can be explained by the inactivation of lysine racemase by Pip2C.

Comparison with Mammalian Pip2C/Pyr2C Reductase—Lysine is metabolized through pipecolic acid in the mammalian brain but through saccharopine in other organs (34–36). The enzymatic activity of Pip2C/Pyr2C reductase has been demonstrated in rat tissues (15), hog kidney (18), brains of dog, monkey, and mouse (17), plants (Phaseolus radiatus and Pisum sativum), and Neurospora crassa (16). The enzyme purified from rat kidney acts on both Pip2C and Pyr2C in the same manner as the P. putida DpkA. However, the rat enzyme uses NADH as effectively as NADPH and catalyzes only the one-way reduction of Pip2C and Pyr2C in contrast to the bacterial enzyme also oxidizing L-pipecolate and L-proline. Moreover, the mammalian enzyme is inhibited by N-formyl-L-phenylalanine (17, 18), whereas the bacterial counterpart is not affected by this compound. No mammalian proteins with significant similarities to the bacterial DpkA have been found by a BLAST search with various protein data bases. Therefore, the mammalian Pip2C/Pyr2C reductase probably belongs to a completely different family of proteins from the bacterial DpkA.


   FOOTNOTES
 
* This work was supported in part by Grant-in-aid for Scientific Research on Priority Areas (B) 13125203 (to N. E.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, by Grant-in-aid for Encouragement of Young Scientists 15780070 (to H. Mihara) from the Japan Society for the Promotion of Science, by the National Project on Protein Structural and Functional Analyses, and by a grant-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (21st Century COE on Kyoto University Alliance for Chemistry). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

§ These authors contributed equally to this work. Back

|| To whom correspondence should be addressed. Tel.: 81-774-38-3240; Fax: 81-774-38-3248; E-mail: esaki{at}scl.kyoto-u.ac.jp.

1 The abbreviations used are: LDH, lactate dehydrogenase; MDH, malate dehydrogenase; Pip2C, {Delta}1-piperideine-2-carboxylate; Pyr2C, {Delta}1-pyrroline-2-carboxylate; Bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HPLC, high pressure liquid chromatography; SLDH, sulfolactate dehydrogenase from M. jannaschi. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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