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

P. N. Black, Q. Zhang

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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 (C(18:1)), 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 ΔfadL 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. Other mutants had levels of long-chain-fatty-acid binding that were either the same, or nearly the same, as those for the wild-type. On the basis of competition experiments, DEPC treatment and the analyses of the mutants, His110 may function in carboxylate binding.

Original languageEnglish (US)
Pages (from-to)389-394
Number of pages6
JournalBiochemical Journal
Volume310
Issue number2
DOIs
StatePublished - Jan 1 1995

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Escherichia coli
Fatty Acids
Membranes
Proteins
Oleic Acid
Diethyl Pyrocarbonate
Histidine
Mutagenesis
Mutation
Proton transfer
Site-Directed Mutagenesis
Alanine
Agar
Protons
Screening
Membrane Proteins
Plasmids
Hot Temperature
Cells
Alcohols

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{e0b22628c43e41e395ca6863cfce192b,
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 (C(18:1)), 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 ΔfadL 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. Other mutants had levels of long-chain-fatty-acid binding that were either the same, or nearly the same, as those for the wild-type. On the basis of competition experiments, DEPC treatment and the analyses of the mutants, His110 may function in carboxylate binding.",
author = "Black, {P. N.} and Q. Zhang",
year = "1995",
month = "1",
day = "1",
doi = "10.1042/bj3100389",
language = "English (US)",
volume = "310",
pages = "389--394",
journal = "Biochemical Journal",
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T1 - 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

T2 - Possible role of this residue in carboxylate binding

AU - Black, P. N.

AU - Zhang, Q.

PY - 1995/1/1

Y1 - 1995/1/1

N2 - 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 (C(18:1)), 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 ΔfadL 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. Other mutants had levels of long-chain-fatty-acid binding that were either the same, or nearly the same, as those for the wild-type. On the basis of competition experiments, DEPC treatment and the analyses of the mutants, His110 may function in carboxylate binding.

AB - 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 (C(18:1)), 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 ΔfadL 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. Other mutants had levels of long-chain-fatty-acid binding that were either the same, or nearly the same, as those for the wild-type. On the basis of competition experiments, DEPC treatment and the analyses of the mutants, His110 may function in carboxylate binding.

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