title: Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids.
abstract: FadD catalyses the first step in E. coli beta-oxidation, the activation of free fatty acids into acyl-CoA thioesters. This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes. Alcohols and alkanes derived from medium chain fatty acids (MCFAs, 6-12 carbons) are potential biofuels; however, FadD has low activity on MCFAs. Herein, we generate mutations in fadD that enhance its acyl-CoA synthetase activity on MCFAs. Homology modeling reveals that these mutations cluster on a face of FadD from which the co-product, AMP, is expected to exit. Using FadD homology models, we design additional FadD mutations that enhance E. coli growth rate on octanoate and provide evidence for a model wherein FadD activity on octanoate can be enhanced by aiding product exit. These studies provide FadD mutants useful for producing MCFA derivatives and a rationale to alter the substrate specificity of adenylating enzymes.
authors.affiliation: Department of Systems Biology, Harvard Medical School , Boston, MA , USA.
authors.forename: Tyler J
authors.initials: TJ
authors.lastname: Ford
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard Medical School , Boston, MA , USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Way
keywords: CoA synthetase; Fatty acid metabolism; Fatty acids; Protein engineering