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 coli, were determined. The deduced amino acid sequence showed homologies with 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 Km values for pyruvate, lactate, NADH and NAD were 0.3, 19, 0.03 and 0.16 mM, indicating that the enzyme reduced pyruvate in vivo. Besides the D-LDH activity, L. mesenteroides subsp. cremoris also displayed HicDH enzymatic activity, catalysing the reduction of pyruvate analogs. The purified D-LDH displayed low HicDH-type activity; therefore, differences in specificity profiles between the crude extract and the purified enzyme suggested the occurrence of a specific HicDH.
Dartois
V
V
ENSBANA, Laboratoire de Microbiologie, Dijon, France.
Phalip
V
V
Schmitt
P
P
Diviès
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D045022:Lactate Dehydrogenases; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D004269:DNA, Bacterial; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase