A New Family of Electron-Bifurcating Enzymes Represented by Ferredoxin- and NAD-Dependent Glycerol-3-Phosphate Oxidoreductase.

Glycerol is abundant in natural environments and is utilized by many microbes. In aerobes, it is typically converted, via glycerol-3-phosphate (G3P), to dihydroxyacetone phosphate (DHAP) by a quinone-linked dehydrogenase thereby coupling glycolysis and respiration. Under anaerobic conditions, an NAD+-dependent dehydrogenase has been proposed to oxidize G3P to DHAP in several bacteria but this reaction is thermodynamically unfavorable. Herein, we have purified a heterotrimeric electron bifurcating enzyme from the archaeon Pyrococcus furiosus where the simultaneous oxidation of both G3P (Eo' -190 mV) and reduced ferredoxin (Em ∼ -450 mV) enables reduction of NAD+(Eo' -320 mV) and production of DHAP. Surprisingly, this ferredoxin-dependent NAD+G3P oxidoreductase (FngABC) is unrelated evolutionarily to the four known families of bifurcating enzyme. Analysis by UV-visible spectroscopy and of its predicted structure suggest FngABC has a nonclassical bifurcation mechanism. Phylogenetic and genome context analyses of 4,273 P. furiosus FngA homologues revealed they are widespread with ∼35% found mainly in Clostridia as FngABC and ∼10% found mainly in Actinomyces as FngAB, most of which utilize polyols in addition to glycerol. The remaining ∼40% are present in Gammaproteobacteria and halophilic archaea but here FngA is associated with homologues of E. coli GlpBC, which are unrelated to FngBC, and these enzymes typically only use glycerol. It is therefore proposed that P. furiosus FngABC and the previously characterized E. coli GlpABC are disparate members of a new electron bifurcating family widespread in the microbial world that are involved in the metabolism of a range of sugars as well as glycerol.