title: Evidence that His110 of the protein FadL in the outer membrane of Escherichia coli is involved in the binding and uptake of long-chain fatty acids: possible role of this residue in carboxylate binding.
abstract: The binding of exogenous fatty acids to the outer-membrane protein FadL of Escherichia coli is specific for long-chain fatty acids (C14-C18). Oleoyl alcohol [(Z)-9-octadecen-1-ol] and methyl oleate were unable to displace FadL-specific binding of [3H]oleate (C18:1), suggesting that the carboxylate of the long-chain fatty acid was required for binding. Therefore the binding of exogenous fatty acids to FadL is governed, in part, by the carboxy group of the long-chain fatty acid. Treatment of whole cells with 1 mM diethyl pyrocarbonate (DEPC) depressed binding by 43-73% over the range of oleate concentrations used (10-500 nM). On the basis of these results and the notion that histidine residues often play a role involving proton transfer and charge-pairing, the five histidine residues within FadL (His110, His226, His327, His345 and His418) were replaced by alanine using site-directed mutagenesis. Altered FadL proteins were correctly localized in the outer membrane at wild-type levels and retained the heat-modifiable property characteristic of the wild-type protein. Initial screening of these fadL mutants revealed that the replacement of His110 by Ala resulted in a decreased growth rate on minimal oleate/agar plates. The rates of long-chain fatty acid transport for delta fadL strains harbouring each mutation on a plasmid, with the exception of fadLH110A, were the same, or nearly the same, as those for the wild-type. fadLH110A was also defective in binding, arguing that the functional effect of this mutation was at the level of long-chain-fatty-acid binding.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Tennessee College of Medicine, Memphis 38163, USA.
authors.forename: P N
authors.initials: PN
authors.lastname: Black
authors.forename: Q
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D050612:Fatty Acid Transport Proteins; D005798:Genes, Bacterial; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D050612:Fatty Acid Transport Proteins; D009829:Oleic Acids; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C065691:fadL protein, E coli; D019301:Oleic Acid; D006639:Histidine; D004047:Diethyl Pyrocarbonate; D000409:Alanine