PURIFICATION, PROPERTIES AND DNA-SEQUENCE OF THE D-LACTATE DEHYDROGENASE FROM LEUCONOSTOC-MESENTEROIDES SUBSP CREMORIS

The complete sequence of the D-lactate dehydrogenase (D-ldh) gene from Leuconostoc mesenteroides subsp. cremoris, cloned in Escherichia coil , were determined. The deduced amino acid sequence showed homologies w ith all members of the D-specific-2-hydroxyacid dehydrogenase family. Furthermore, the essential residues detected so far as being involved in catalysis were also conserved. Purification of the enzyme revealed physico-chemical properties corresponding to those predicted from the sequence. The active enzyme was a dimer of 40-kDa subunits. The K-m va lues for pyruvate, lactate, NADH and NAD were 0.3, 19, 0.03 and 0.16 m M, indicating that the enzyme reduced pyruvate in vivo. Besides the D- LDH activity, L. mesenteroides subsp. cremoris also displayed HicDH en zymatic activity, catalysing the reduction of pyruvate analogs. The pu rified D-LDH displayed low HicDH-type activity; therefore, differences in specificity profiles between the crude extract and the purified en zyme suggested the occurrence of a specific HicDH.