Direct coupling of lactate oxidation with butyryl-CoA formation via a canonical electron transfer flavoprotein in Fusobacterium nucleatum

The gram-negative opportunistic pathogen Fusobacterium nucleatum encodes an electron transfer flavoprotein (ETF) within a 6-gene cluster that also includes genes for a D-lactate dehydrogenase (Ldh), butyryl-CoA dehydrogenase (Bcd), and LrgAB. Herein, we demonstrate that ETF functions as a canonical ETF, transferring two electrons from Ldh following oxidation of D-lactate to Bcd for the reduction of crotonyl-CoA to butyryl-CoA. Steady-state kinetic analysis of the LdhFN/ETFFN/BcdFN reaction (lactate + crotonyl-CoA → pyruvate + butyryl-CoA) yielded a kcat of 2.5 ± 0.1 s-1 and a KM of 0.65 ± 0.04 μM and 5.2 ± 0.5 μM for D-lactate and butyryl-CoA, respectively. As observed in homologous ETFs, the FAD cofactor of ETF forms the red anionic semiquinone (FAD•-) but the Eo' values (versus the normal hydrogen electrode) of -70 mV (FAD/FAD•-) and = -122 mV (FAD•-/FADH-) are more compressed and negative compared to other ETFs, indicating the flavoprotein is physiologically primed to accept two electrons from Ldh. Similarly, reductive titration of Ldh shows that its FAD cofactor also forms the red anionic semiquinone, but the Eo' values for FAD/FAD•- (-109 mV) and FAD•-/FADH- (-115 mV) are even more closely spaced. We discuss how F. nucleatum potentially uses this lactate utilization gene cluster to maintain redox homeostasis during oxidative stress and how beneficial gut anaerobes of the Lachnospiraceae family with similar gene clusters employ either a canonical or bifurcating ETF for the conversion of lactate (and acetate) to butyrate.