Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids: Amino acid residues within the ATP/AMP signature motif of Escherichia coli fadD are required for enzyme activity and fatty acid transport

James D. Weimar, Concetta C. DiRusso, Raymond Delio, Paul N. Black

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Abstract

Fatty acyl-CoA synthetase (FACS, fatty acidtCoA ligase, AMP forming; EC 6.2.1.3) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. In Escherichia coli this enzyme, encoded by the fadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids. The E. coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif (213YTGGTTGVAKGA 224 and 356GYGLTE361) placing it in the superfamily of adenylate-forming enzymes. A series of site-directed mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport. This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25-45% wild type activity (fadD G21GA, fadDT217A, fadDG219A, and fadD K222) and 2) those with 10% or less wild-type activity (fadD Y213A, fadDT214A, and fadDE361A). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.

Original languageEnglish (US)
Pages (from-to)29369-29376
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number33
DOIs
StatePublished - Aug 16 2002

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Coenzyme A Ligases
Enzyme activity
Adenosine Monophosphate
Acyl Coenzyme A
Escherichia coli
Fatty Acids
Adenosine Triphosphate
Chemical activation
Amino Acids
Enzymes
Genes
Mutation
Cell membranes
Ligases
Metabolism
Esters
Western Blotting
Cell Membrane
Phenotype
Defects

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids: Amino acid residues within the ATP/AMP signature motif of Escherichia coli fadD are required for enzyme activity and fatty acid transport",
abstract = "Fatty acyl-CoA synthetase (FACS, fatty acidtCoA ligase, AMP forming; EC 6.2.1.3) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. In Escherichia coli this enzyme, encoded by the fadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids. The E. coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif (213YTGGTTGVAKGA 224 and 356GYGLTE361) placing it in the superfamily of adenylate-forming enzymes. A series of site-directed mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport. This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25-45{\%} wild type activity (fadD G21GA, fadDT217A, fadDG219A, and fadD K222) and 2) those with 10{\%} or less wild-type activity (fadD Y213A, fadDT214A, and fadDE361A). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.",
author = "Weimar, {James D.} and DiRusso, {Concetta C.} and Raymond Delio and Black, {Paul N.}",
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T1 - Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids

T2 - Amino acid residues within the ATP/AMP signature motif of Escherichia coli fadD are required for enzyme activity and fatty acid transport

AU - Weimar, James D.

AU - DiRusso, Concetta C.

AU - Delio, Raymond

AU - Black, Paul N.

PY - 2002/8/16

Y1 - 2002/8/16

N2 - Fatty acyl-CoA synthetase (FACS, fatty acidtCoA ligase, AMP forming; EC 6.2.1.3) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. In Escherichia coli this enzyme, encoded by the fadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids. The E. coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif (213YTGGTTGVAKGA 224 and 356GYGLTE361) placing it in the superfamily of adenylate-forming enzymes. A series of site-directed mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport. This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25-45% wild type activity (fadD G21GA, fadDT217A, fadDG219A, and fadD K222) and 2) those with 10% or less wild-type activity (fadD Y213A, fadDT214A, and fadDE361A). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.

AB - Fatty acyl-CoA synthetase (FACS, fatty acidtCoA ligase, AMP forming; EC 6.2.1.3) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. In Escherichia coli this enzyme, encoded by the fadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids. The E. coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif (213YTGGTTGVAKGA 224 and 356GYGLTE361) placing it in the superfamily of adenylate-forming enzymes. A series of site-directed mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport. This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25-45% wild type activity (fadD G21GA, fadDT217A, fadDG219A, and fadD K222) and 2) those with 10% or less wild-type activity (fadD Y213A, fadDT214A, and fadDE361A). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.

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