The Archaeoglobus fulgidus D-lactate dehydrogenase is a Zn2+ flavoprotein

Archaeoglobus fulgidus, a hyperthermophilic, archaeal sulfate reducer, is o ne of the few organisms that can utilize D-lactate as a sole source for bot h carbon and electrons. The A. fulgidus open reading frame, AF0394, which i s predicted to encode a D-(-)-lactate dehydrogenase (Dld), was cloned, and its product was expressed in Escherichia coli as a fusion with the maltose binding protein (MBP), The 90-kDa MBP-Dld fusion protein was more efficient ly expressed in E, coli when coexpressed with the E. coli dnaY gene, encodi ng the arginyl tRNA for the codons AGA and AGG. When cleaved from the fusio n protein by treatment with factor Xa, the recombinant Dld (rDld) has an ap parent molecular mass of 50 kDa, similar to that of the native A. fulgidus Bid enzyme. Both the purified MBP-Dld fusion protein and its rDld cleavage fragment have lactate dehydrogenase activities specific for D-lactate, are stable at 80 degrees C, and retain activity after exposure to oxygen, The f lavin cofactor FAD, which binds rDld apoprotein with a 1:1 stoichiometry, i s essential for activity.