Catalytic, Computational, and Evolutionary Analysis of the d‑Lactate Dehydrogenases Responsible for d‑Lactic Acid Production in Lactic Acid Bacteria

d-Lactate dehydrogenase (d-LDH) catalyzes the
reversible reaction pyruvate + NADH + H+ ↔ lactate
+ NAD+, which is a principal step in the production of d-lactate in lactic acid bacteria. In this study, we identified
and characterized the major d-LDH (d-LDH1) from
three d-LDHs in Leuconostoc mesenteroides, which has been extensively used in food processing. A molecular
simulation study of d-LDH1 showed that the conformation changes
during substrate binding. During catalysis, Tyr101 and Arg235 bind
the substrates by hydrogen bonds and His296 acts as a general acid/base
for proton transfer. These residues are also highly conserved and
have coevolved. Point mutations proved that the substrate binding
sites and catalytic site are crucial for enzyme activity. Network
and phylogenetic analyses indicated that d-LDH1 and the homologues
are widely distributed but are most abundant in bacteria and fungi.
This study expands the understanding of the functions, catalytic mechanism,
and evolution of d-LDH.