N‐Methyl‐l‐amino acid dehydrogenase from Pseudomonas putida

Abstract

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.

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

“…PsDpkA specifically acted on methylamine and not on ammonia. Therefore, the substrate specificities of PsDpkA toward ␣-keto acids and amines were similar to those of PpDpkA (24). The substrate specificity of the enzyme in the NADP ϩ -dependent oxidative reaction was also similar to that of PpDpkA (data not shown) (24).…”

“…Therefore, the substrate specificities of PsDpkA toward ␣-keto acids and amines were similar to those of PpDpkA (24). The substrate specificity of the enzyme in the NADP ϩ -dependent oxidative reaction was also similar to that of PpDpkA (data not shown) (24). PsDpkA showed its maximum activity between 30 and 45°C and was stable between 0 and 35°C after a 30-min incubation in 20 mM Tris-HCl buffer at pH 7.0.…”

“…Alcohol dehydrogenases with the Rossmann fold require a metal ion such as Zn 2ϩ bound to the active site to stabilize a transition state such as an alkoxide (43,44). The catalytic reaction of PsDpkA is not accelerated but inhibited by metal ions (24), and no metal ion bound to the protein was detected on the residual electron density map. On the basis of geometrical considerations, the predicted catalytic mechanism of Pip2C/Pyr2C reductase is shown in Fig.…”

“…A BLAST search of the P. syringae genome sequence using the PpDpkA (DpkA from P. putida ATCC12633) sequence identified a putative protein, PSPTO2359 (NCBI database accession AAO55870), which has been annotated as UGDH encoded by the allD gene (36). However, our recent sequence analysis indicated that the annotation for PSPTO2359 is not correct, and we have provided a revised annotation for the gene product as a DpkA protein, which is a multifunctional enzyme with activities of Pip2C/Pyr2C reductase (8) and N-methyl-L-amino acid dehydrogenase (24). The amino acid sequence of P. syringae DpkA shows a 71% sequence identity with that of PpDpkA.…”

“…Enzyme Assays-N-Methyl-L-amino acid dehydrogenase assays were carried out by measuring the decrease in the amount of NADPH at 340 nm with an MPS-2000 spectrophotometer (Shimadzu, Kyoto, Japan) at 30°C (24). A standard reaction mixture contained 10 mM pyruvate, 60 mM methylamine-H 2 SO 4 (pH 10.0), 0.2 mM NADPH, and the enzyme in a final volume of 1 ml.…”