Characterization of FadL-specific fatty acid binding in Escherichia coli

Research output: Contribution to journalArticle

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Abstract

The product of the fadL gene (FadL) is a central component of the long-chain fatty acid transport system of Escherichia coli. When fatty acid activation is blocked by a mutation in the structural gene for acyl CoA synthetase (fadD) transport is inhibited allowing a FadL-specific fatty acid binding activity to be measured. This binding activity was 4- to 6-fold greater in the fadL+fadD strain LS6928 when compared to the Δ fadL fadD strain LS6929. With long-chain fatty acids, this binding activity was saturable and it was estimated that there were approx. 35 000 FadL-specific oleic acid binding sites per cell in the fadL + strain LS6928. The FadL-specific fatty acid binding affinity was highest for oleic acid (18:1) and palmitic acid (16:0) giving apparent KD values of 2.3 · 10-7 M and 8.8 · 10-7 M, respectively. FadL-specific binding affinity of myristic acid (14:0) was nearly an order of magnitude less and no FadL-specific binding of decanoic acid (10:0) could be measured. Two lines of evidence suggest that FadL-fatty acid binding occurs by a hydrophobic interaction: (1) There was a preference for the long-chain substrates oleic acid and palmitic acid; and (2) oleic acid binding activity was not significantly changed over the pH range 5.0 to 8.0. The FadL-specific binding of oleic acid in the fadL + strain LS6928 could be blocked by preincubarion with antisera raised against purified FadL providing a clear correlation between the activity and identity of FadL. The binding activity associated with FadL was measured in vesicles of the outer membrane following passage over the hydrophobic resin Lipidex 1000. The KD of oleic acid binding attributable to FadL in outer membrane vesicles (6.0 · 10-7 M) was in close agreement with that determined in whole cells. Overall, these studies demonstrated that FadL binds long-chain fatty acids with a relatively high affinity prior to their transport across the outer membrane.

Original languageEnglish (US)
Pages (from-to)97-105
Number of pages9
JournalBiochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
Volume1046
Issue number1
DOIs
StatePublished - Aug 28 1990

Fingerprint

Escherichia coli
Oleic Acid
Fatty Acids
Palmitic Acid
Membranes
Genes
Coenzyme A Ligases
Myristic Acid
Hydrophobic and Hydrophilic Interactions
Immune Sera
Resins
Chemical activation
Binding Sites
Mutation
Substrates

Keywords

  • (E. coli)
  • Fatty acid binding protein
  • Fatty acid transfer
  • Fatty acid transport

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Endocrinology

Cite this

Characterization of FadL-specific fatty acid binding in Escherichia coli. / Black, Paul N.

In: Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism, Vol. 1046, No. 1, 28.08.1990, p. 97-105.

Research output: Contribution to journalArticle

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AB - The product of the fadL gene (FadL) is a central component of the long-chain fatty acid transport system of Escherichia coli. When fatty acid activation is blocked by a mutation in the structural gene for acyl CoA synthetase (fadD) transport is inhibited allowing a FadL-specific fatty acid binding activity to be measured. This binding activity was 4- to 6-fold greater in the fadL+fadD strain LS6928 when compared to the Δ fadL fadD strain LS6929. With long-chain fatty acids, this binding activity was saturable and it was estimated that there were approx. 35 000 FadL-specific oleic acid binding sites per cell in the fadL + strain LS6928. The FadL-specific fatty acid binding affinity was highest for oleic acid (18:1) and palmitic acid (16:0) giving apparent KD values of 2.3 · 10-7 M and 8.8 · 10-7 M, respectively. FadL-specific binding affinity of myristic acid (14:0) was nearly an order of magnitude less and no FadL-specific binding of decanoic acid (10:0) could be measured. Two lines of evidence suggest that FadL-fatty acid binding occurs by a hydrophobic interaction: (1) There was a preference for the long-chain substrates oleic acid and palmitic acid; and (2) oleic acid binding activity was not significantly changed over the pH range 5.0 to 8.0. The FadL-specific binding of oleic acid in the fadL + strain LS6928 could be blocked by preincubarion with antisera raised against purified FadL providing a clear correlation between the activity and identity of FadL. The binding activity associated with FadL was measured in vesicles of the outer membrane following passage over the hydrophobic resin Lipidex 1000. The KD of oleic acid binding attributable to FadL in outer membrane vesicles (6.0 · 10-7 M) was in close agreement with that determined in whole cells. Overall, these studies demonstrated that FadL binds long-chain fatty acids with a relatively high affinity prior to their transport across the outer membrane.

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