title: Dissection and engineering of the Escherichia coli formate hydrogenlyase complex.
abstract: The Escherichia coli formate hydrogenlyase (FHL) complex is produced under fermentative conditions and couples formate oxidation to hydrogen production. In this work, the architecture of FHL has been probed by analysing affinity-tagged complexes from various genetic backgrounds. In a successful attempt to stabilize the complex, a strain encoding a fusion between FdhF and HycB has been engineered and characterised. Finally, site-directed mutagenesis of the hycG gene was performed, which is predicted to encode a hydrogenase subunit important for regulating sensitivity to oxygen. This work helps to define the core components of FHL and provides solutions to improving the stability of the enzyme.
authors.affiliation: School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: McDowall
authors.affiliation: School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: M Charlotte
authors.initials: MC
authors.lastname: Hjersing
authors.affiliation: School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
authors.forename: Tracy
authors.initials: T
authors.lastname: Palmer
authors.affiliation: School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK. Electronic address: f.sargent@dundee.ac.uk.
authors.forename: Frank
authors.initials: F
authors.lastname: Sargent
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering; D006864:Hydrogenase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009876:Operon / Q000235:genetics; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase; D010100:Oxygen
keywords: Escherichia coli; Fermentation; Formate hydrogenlyase; Genetic engineering; Mutagenesis; [NiFe]-hydrogenase