NAD-dependent lactate dehydrogenase catalyses the first step in respiratory utilization of lactate by Lactococcus lactis

• LutABC proteins do not participate in lactate oxidation in Lactococcus lactis
• Lactococcus lactis has very low NAD-independent lactate dehydrogenase activity
• Fructose-1,6-bisphosphate-dependent lactate dehydrogenase can work in reverse in vivo
• Metabolite concentrations in the stationary phase are favorable for lactate oxidation
• Respiratory metabolism is the basis for continual lactate oxidation in Lactococcus

Lactococcus lactis can undergo respiration when hemin is added to an aerobic culture. The most distinctive feature of lactococcal respiration is that lactate could be consumed in the stationary phase concomitantly with the rapid accumulation of diacetyl and acetoin. However, the enzyme responsible for lactate utilization in this process has not yet been identified. As genes for fermentative NAD-dependent l-lactate dehydrogenase (l-nLDH) and potential electron transport chain (ETC)-related NAD-independent l-LDH (l-iLDH) exist in L. lactis, the activities of these enzymes were measured in this study using crude cell extracts prepared from respiratory and fermentation cultures. Further studies were conducted with purified preparations of recombinant LDH homologous proteins. The results showed that l-iLDH activity was hardly detected in both crude cell extracts and purified l-iLDH homologous protein while l-nLDH activity was very significant. This suggested that l-iLDHs were inactive in lactate utilization. The results of kinetic analyses and the effects of activator, inhibitor, substrate and product concentrations on the reaction equilibrium showed that l-nLDH was much more prone to catalyze the pyruvate reduction reaction but could reverse its role provided that the concentrations of NADH and pyruvate were extremely low while NAD and lactate were abundant. Metabolite analysis in respiratory culture revealed that the cellular status in the stationary phase was beneficial for l-nLDH to catalyze lactate oxidation. The factors accounting for the respiration- and stationary phase-dependent lactate utilization in L. lactis are discussed here.