Lactate dehydrogenase has been extensively studied for a long time. At present, it is well known that two isomeric forms of lactic acid, L- and D-lactic acids, are produced by two distinct stereospecific NAD-dependent lactate dehydrogenases (nLDHs), i.e., L-lactate dehydrogenase (L-ldh) and D-lactate dehydrogenase (D-ldh), respectively.

In this study, D-lactate dehydrogenase gene was amplified from S. inulinus CASD and the enzyme (D-lactic acid dehydrogenase, DLDH744) was purified from E. coli. DLDH744 is a homodimeric protein with the subunit molecular weight of 37 kDa. The reaction product catalyzed by DLDH744 was only D-lactic acid from pyruvate without any L-lactic acid detected. Both NADH and NADPH could serve as the coenzyme, which is different feature compared to other reported Lactate dehydrogenases. The Km values for NADH and NADPH were 0.086±0.001 and 0.072±0.000 mM, respectively. Furthermore, the activity of glutamate dehydrogenase with the ability to produce α-ketoglutarate from glutamic acid with NAD as coenzyme was also confirmed. To investigate the catalytic and coenzyme binding mechanism, the crystal structure of the enzyme DLDH744 was demonstrated. And several key amino acids were confirmed, which are responsible for catalyst and coenzyme binding.