Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

Abstract

c Moorella thermoacetica can grow with H 2 and CO 2 , forming acetic acid from 2 CO 2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown.

Energy Conservation Associated with Ethanol Formation from H 2 and CO 2 in Clostridium autoethanogenum Involving Electron Bifurcation

“…This is in contrast to Moorella thermoacetica, in which one of three formate dehydrogenases is predicted to have a "periplasmic" location (52,54,62).…”

“…Our results indicate that in C. autoethanogenum, a close relative of C. ljungdahlii, ferredoxin and NADP are the electron acceptors for H 2 oxidation (reaction 3) and that CO 2 is reduced with H 2 in a reaction that does not involve ferredoxin and/or NAD(P)H. But the third open question, whether or not the methylene-H 4 F reductase is electron bifurcating and "reduces ferredoxin," remains to be answered. In Moorella thermoacetica, there is indirect evidence for an electron-bifurcating and NAD-specific methylene-H 4 F reductase (62).…”

“…Bioinformatic analyses indicated that MetF lacks an NAD(P) binding site and favors FMN as a cofactor. MetF exhibited high methylene-H 4 F reductase activity with benzyl viologen only when produced together with the iron-sulfur zinc protein MetV, which in part shows sequence similarity to MetF (23,62). The nature of the electron donor(s) is still uncertain (see Discussion).…”

“…In these complexes EtfAB is assumed to be the site of electron bifurcation (38,70). (12) MetFV alone catalyzes only the reduction of methylene-H 4 F with reduced viologen dyes (62), and EtfAB alone only catalyzes the reduction of viologen dyes with NADH (70). Both activities are found in cell extracts of C. autoethanogenum but a ferredoxindependent reduction of methylene-H 4 F with NADH (reaction 12) or NADPH has not yet been demonstrated, maybe because MetFV and EtfAB only form a loose complex which dissociates in the cell extracts.…”

Heterotrimeric NADH-Oxidizing Methylenetetrahydrofolate Reductase from the Acetogenic Bacterium Acetobacterium woodii

“…Constructs were verified via DNA sequencing and introduced into E. coli C41(DE3), which already carried plasmids pRKISC (24) and pCodonPlus, for expression. For expression, cells were grown according to a previously described method (25). The cells were harvested, washed with lysis buffer A (50 mM NaH 2 PO 4 , 10 mM imidazole, 20% [vol/vol] glycerol, 300 mM NaCl, 10 M [each] flavin mononucleotides [FMN], 2 mM DTE, pH 8), and used immediately for purification.…”