NADPH-specific meso-diaminopimelate dehydrogenase from the archaeon Pyrococcus sp. stereoselectively aminates α-keto acids using NADH

Enzymatic asymmetric D-amination of carbonyl compounds and keto acids is of great interest in biotechnology. Stable D-stereoselective NAD-dependent dehydrogenases are required for the synthesis of D-amino acids from α-keto acids, because they can use ammonium ion as the amino group donor and allow high product yield via coenzyme recycling. However, these enzymes are rare in nature. Here, we describe the first archaeal meso-diaminopimelate dehydrogenase from Pyrococcus sp. (PyrDAPDH). The enzyme is active with either NADP(H) or NAD(H) and catalyzes reductive amination of various α-keto acids and oxidative deamination of DAP and D-alanine. At 60 °C, the rates of pyruvate and phenylpyruvate amination reach 220 ± 20 and 8.5 ± 0.3 U/mg, respectively. Using X-ray analysis, we suggested structural determinants supporting the specificity of PyrDAPDH for both NADPH and NADH. Structure-based site-directed mutagenesis helped to achieve an 83% yield of D-phenylglycine in the 24-hour reductive amination of phenylglyoxylate with 1 mM NAD(H) and 1.0 M NH4Cl at 40 °C. By changing active site residues, we revealed a key role for aspartate (D101) but not histidine (H163) in the amination of α-keto acids. The obtained results demonstrate the suitability of dehydrogenases for asymmetric D-amination, show the advantages of enzymes from Archaea for biotechnological applications, and deepen our knowledge of structure-function relationships in diaminopimelate dehydrogenases.