NADP+-Preferring D-Lactate Dehydrogenase from Sporolactobacillus inulinus

Hydroxyacid dehydrogenases, including L- and D-lactate dehydrogenase (LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and have a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD⁺ as cofactor. The LDH that can effectively use NADPH as a coenzyme could be a good alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a D-lactate dehydrogenase (D-LDH) from a Sporolactobacillus inulinus strain was experimentally determined to use both NADH and NADPH with high efficiencies and with a preference for NADPH as coenzyme, which is different from all the previously reported LDHs in coenzyme utilization. The enzyme was also found to have both the D-LDH and glutamate dehydrogenase (GDH) activities. Then the biochemical properties of the enzyme D-LDH were illustrated by X-ray crystal structural characterization, in vivo and in vitro enzymatic activity analysis. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.