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)...

We found N‐methyl‐l‐amino acid dehydrogenase activity in various bacterial strains, such as Pseudomonas putida and Bacillus alvei, and cloned the gene from P. putida ATCC12633 into Escherichia coli. The enzyme purified to homogeneity from recombinant E. coli catalyzed the NADPH‐dependent formation of N‐alkyl‐l‐amino acids from the corresponding α‐oxo acids (e.g. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (e.g. methylamine, ethylamine, and propylamine). Ammonia was inert as a substrate, and the enzyme was clearly distinct from conventional NAD(P)‐dependent amino acid dehydrogenases, such as alanine dehydrogenase (EC 1.4.1.1). NADPH was more than 300 times more efficient than NADH as a hydrogen donor in the enzymatic reductive amination. Primary structure analysis revealed that the enzyme belongs to a new NAD(P)‐dependent oxidoreductase superfamily, the members of which show no sequence homology to conventional NAD(P)‐dependent amino acid dehydrogenases and opine dehydrogenases.

In the Mallik 2002 Gas Hydrate Production Research Well Program, formation tests with a Modular Formation Dynamics Tester (MDT)TM tool were conducted and the test results were analyzed using conventional pressure-transient test-analysis methods. The reliability of the reservoir-parameter estimates, however, is uncertain because of the abrupt change in gas hydrate saturation associated with gas hydrate dissociation during the tests. To investigate the applicability of these methods, the bottomhole-pressure responses during MDT tests in the hypothetical and actual gas hydrate zones were predicted using a numerical simulator and then analyzed by conventional test-analysis methods. This study revealed that the conventional methods might indicate the average effective permeability over the area of gas hydrate dissociation, and that they might accurately suggest the radius of gas hydrate dissociation only when applying appropriate multiphase-fluid properties and production rates to the cases with high gas hydrate saturation.

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