d-lactate dehydrogenases are known to be expressed by prokaryotes and by eukaryotic invertebrates, and over the years the functional and structural features of some bacterial representatives of this enzyme ensemble have been investigated quite in detail. Remarkably, a human gene coding for a putative d-lactate dehydrogenase (DLDH) was identified and characterized, disclosing the occurrence of alternative splicing of its primary transcript. This translates into the expression of two human DLDH (hDLDH) isoforms, the molecular mass of which is expected to differ by 2.7 kDa. However, no information on these two hDLDH isoforms is available at the protein level. Here we report on the catalytic action of these enzyme... More
d-lactate dehydrogenases are known to be expressed by prokaryotes and by eukaryotic invertebrates, and over the years the functional and structural features of some bacterial representatives of this enzyme ensemble have been investigated quite in detail. Remarkably, a human gene coding for a putative d-lactate dehydrogenase (DLDH) was identified and characterized, disclosing the occurrence of alternative splicing of its primary transcript. This translates into the expression of two human DLDH (hDLDH) isoforms, the molecular mass of which is expected to differ by 2.7 kDa. However, no information on these two hDLDH isoforms is available at the protein level. Here we report on the catalytic action of these enzymes, along with a first analysis of their structural features. In particular, we show that hDLDH is strictly stereospecific, with the larger isoform (hDLDH-1) featuring higher activity at the expense of d-lactate when compared to its smaller counterpart (hDLDH-2). Furthermore, we found that hDLDH is strongly inhibited by oxalate, as indicated by a K equal to 1.2 μM for this dicarboxylic acid. Structurally speaking, hDLDH-1 and hDLDH-2 were determined, by means of gel filtration and dynamic light scattering experiments, to be a hexamer and a tetramer, respectively. Moreover, in agreement with previous studies performed with human mitochondria, we identified FAD as the cofactor of hDLDH, and we report here a model of FAD binding by the human d-lactate dehydrogenase. Interestingly, the mutations W323C and T412 M negatively affect the activity of hDLDH, most likely by impairing the enzyme electron-acceptor site.
