The Putative Malate/Lactate Dehydrogenase from Pseudomonas putida Is an NADPH-dependent  1 -Piperideine-2-carboxylate/ 1 - Pyrroline-2-carboxylate Reductase Involved in the Catabolism of D-Lysine and D-Proline* □ S Received for publication, October 20, 2004, and in revised form, November 22, 2004 Published, JBC Papers in Press, November 23, 2004, DOI 10.1074/jbc.M411918200 Hisashi Muramatsu‡§, Hisaaki Mihara‡§, Ryo Kakutani‡, Mari Yasuda¶, Makoto Ueda¶, Tatsuo Kurihara‡, and Nobuyoshi Esaki‡ From the ‡Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan and the ¶Yokohama Research Center, Mitsubishi Chemical Corporation, Yokohama 227-8502, Japan A Pseudomonas putida ATCC12633 gene, dpkA, encod- ing a putative protein annotated as malate/L-lactate de- hydrogenase in various sequence data bases was dis- rupted 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 dehy- drogenase activity but catalyzed the NADPH-dependent reduction of such cyclic imines as  1 -piperideine-2-car- boxylate and  1 -pyrroline-2-carboxylate to form L-pipe- colate and L-proline, respectively. NADH also served as a hydrogen donor for both substrates, although the reac- tion 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 meta- bolic functions, and we named the enzyme  1 -piperi- deine-2-carboxylate/ 1 -pyrroline-2-carboxylate reduc- tase, 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 eu- karyotes (1). They catalyze NAD(P)-dependent interconver- sions between lactate and pyruvate and between malate and oxaloacetate, respectively. However, a new class of NAD(P)-de- pendent malate/L-lactate dehydrogenases with no sequence ho- mology to “orthodox” MDH or LDH has been demonstrated (2, 3). Moreover, they have no Rossmann fold, which is a six- stranded 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/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 mem- bers 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 hypo- thetical 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) de- pendence is common to them, but no other functional similar- ities can be assigned among them. Thus, we expect the occur- rence 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 catab- olized by Pseudomonas putida through the -aminovalerate pathway (11), whereas D-lysine is catabolized through the pipe- colate pathway involving a series of reactions through six-carbon cyclic intermediates (12, 13) (Fig. 1).  1 -Piperideine-2-carboxy- late (Pip2C) is reduced to L-pipecolate by Pip2C reductase (EC 1.5.1.21), which has been purified and characterized by Payton and Chang (14). The reduction of Pip2C is also catalyzed by  1 -pyrroline-2-carboxylate (Pyr2C) reductase (EC 1.5.1.1), which inherently acts on Pyr2C, a five-membered ring homolog of Pip2C, to form L-proline in mammals, plants, and microorgan- isms (15–18). However, it is not yet clear whether such a dual function of Pyr2C reductase is caused by fortuitously broad spec- ificity of the enzyme or of physiological significance. We searched for a new enzyme catalyzing the formation of N-methyl-L-phenylalanine from phenylpyruvate and methyla- mine and found that the enzyme gene is identical to the gene annotated as MDH of the new family of NAD(P)-dependent de- hydrogenases 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 * 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 Alli- ance for Chemistry). The costs of publication of this article were de- frayed 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. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. § These authors contributed equally to this work.  To whom correspondence should be addressed. Tel.: 81-774-38-3240; Fax: 81-774-38-3248; E-mail: esaki@scl.kyoto-u.ac.jp. 1 The abbreviations used are: LDH, lactate dehydrogenase; MDH, malate dehydrogenase; Pip2C,  1 -piperideine-2-carboxylate; Pyr2C,  1 - pyrroline-2-carboxylate; Bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hy- droxymethyl)propane-1,3-diol; HPLC, high pressure liquid chromatog- raphy; SLDH, sulfolactate dehydrogenase from M. jannaschi. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 7, Issue of February 18, pp. 5329 –5335, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 5329 by guest on May 21, 2020 http://www.jbc.org/ Downloaded from