Abstract
Escherichia coli can perform two modes of formate metabolism. Under respiratory conditions, two periplasmically-located formate dehydrogenase isoenzymes couple formate oxidation to the generation of a transmembrane electrochemical gradient; and under fermentative conditions a third cytoplasmic isoenzyme is involved in the disproportionation of formate to CO2 and H2. The respiratory formate dehydrogenases are redox enzymes that comprise three subunits: a molybdenum cofactor- and FeS cluster-containing catalytic subunit; an electron-transferring ferredoxin; and a membrane-integral cytochrome b. The catalytic subunit and its ferredoxin partner are targeted to the periplasm as a complex by the twin-arginine transport (Tat) pathway. Biosynthesis of these enzymes is under control of an accessory protein termed FdhE. In this study, it is shown that E. coli FdhE interacts with the catalytic subunits of the respiratory formate dehydrogenases. Purification of recombinant FdhE demonstrates the protein is an iron-binding rubredoxin that can adopt monomeric and homodimeric forms. Bacterial two-hybrid analysis suggests the homodimer form of FdhE is stabilized by anaerobiosis. Site-directed mutagenesis shows that conserved cysteine motifs are essential for the physiological activity of the FdhE protein and are also involved in iron ligation.
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Abbreviations
- DMSO:
-
Dimethyl sulphoxide
- EPR:
-
Electron paramagnetic resonance
- SPA:
-
Sequential peptide affinity
- Tat:
-
Twin-arginine translocation pathway
- TMAO:
-
Trimethylamine N-oxide
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Acknowledgments
M. G. Hicks (John Innes Centre, Norwich) is thanked for performing some preliminary enzyme assays and W. Yang, X. Yang, S. Chandran and P. Wong (University of Toronto) are thanked for technical assistance. This work was funded in the UK by the BBSRC through awards BB/C006844/2 and BBS/S/P/2003/10319 (to FS) and a grant-in-aid to the John Innes Centre, Norwich. TP is an MRC Senior Non-Clinical Research Fellow and FS is a Royal Society University Research Fellow.
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Communicated by Theo Hansen.
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Lüke, I., Butland, G., Moore, K. et al. Biosynthesis of the respiratory formate dehydrogenases from Escherichia coli: characterization of the FdhE protein. Arch Microbiol 190, 685–696 (2008). https://doi.org/10.1007/s00203-008-0420-4
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DOI: https://doi.org/10.1007/s00203-008-0420-4