Mutational analysis of a fatty acyl-coenzyme a synthetase signature motif identifies seven amino acid residues that modulate fatty acid substrate specificity

Paul N. Black, Qing Zhang, James D. Weimar, Concetta C. DiRusso

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124 Citations (Scopus)

Abstract

Fatty acyl-CoA synthetase (fatty acid:CoA ligase, AMP-forming; EC 6.2.1.3) catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of pyrophosphate. In Escherichia coli this enzyme plays a pivotal role in the uptake of long chain fatty acids (C12- C18) and in the regulation of the global transcriptional regulator FadR. The E. coli fatty acyl-CoA synthetase has remarkable amino acid similarities and identities to the family of both prokaryotic and eukaryotic fatty acyl-CoA synthetases, indicating a common ancestry. Most notable in this regard is a 25-amino acid consensus sequence, DGWLHTGDIGXWX-PXGXLKIIDRKK, common to all fatty acyl-CoA synthetases for which sequence information is available. Within this consensus are 8 invariant and 13 highly conserved amino acid residues in the 12 fatty acyl-CoA synthetases compared. We propose that this sequence represents the fatty acyl-CoA synthetase signature motif (FACS signature motif). This region of fatty acyl-CoA synthetase from E. coli, 431NGWLHTGDIAVMDEEGFL-RIVDRKK 455, contains 17 amino acid residues that are either identical or highly conserved to the FACS signature motif. Eighteen site-directed mutations within the fatty acyl-CoA synthetase structural gene (fadD) corresponding to this motif were constructed to evaluate the contribution of this region of the enzyme to catalytic activity. Three distinct classes of mutations were identified on the basis of growth characteristics on fatty acids, enzymatic activities using cell extracts, and studies using purified wild-type and mutant forms of the enzyme: 1) those that resulted in either wild-type or nearly wild-type fatty acyl-CoA synthetase activity profiles; 2) those that had little or no enzyme activity; and 3) those that resulted in lowering and altering fatty acid chain length specificity. Among the 18 mutants characterized, 7 fall in the third class. We propose that the FACS signature motif is essential for catalytic activity and functions in part to promote fatty acid chain length specificity and thus may compose part of the fatty acid binding site within the enzyme.

Original languageEnglish (US)
Pages (from-to)4896-4903
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number8
DOIs
StatePublished - Feb 21 1997

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Coenzyme A Ligases
Acyl Coenzyme A
Coenzymes
Ligases
Substrate Specificity
Fatty Acids
Amino Acids
Substrates
Escherichia coli
Enzymes
Chain length
Catalyst activity
Mutation
Consensus Sequence
Enzyme activity
Adenosine Monophosphate
Coenzyme A
Cell Extracts
Amino Acid Sequence
Hydrolysis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Mutational analysis of a fatty acyl-coenzyme a synthetase signature motif identifies seven amino acid residues that modulate fatty acid substrate specificity",
abstract = "Fatty acyl-CoA synthetase (fatty acid:CoA ligase, AMP-forming; EC 6.2.1.3) catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of pyrophosphate. In Escherichia coli this enzyme plays a pivotal role in the uptake of long chain fatty acids (C12- C18) and in the regulation of the global transcriptional regulator FadR. The E. coli fatty acyl-CoA synthetase has remarkable amino acid similarities and identities to the family of both prokaryotic and eukaryotic fatty acyl-CoA synthetases, indicating a common ancestry. Most notable in this regard is a 25-amino acid consensus sequence, DGWLHTGDIGXWX-PXGXLKIIDRKK, common to all fatty acyl-CoA synthetases for which sequence information is available. Within this consensus are 8 invariant and 13 highly conserved amino acid residues in the 12 fatty acyl-CoA synthetases compared. We propose that this sequence represents the fatty acyl-CoA synthetase signature motif (FACS signature motif). This region of fatty acyl-CoA synthetase from E. coli, 431NGWLHTGDIAVMDEEGFL-RIVDRKK 455, contains 17 amino acid residues that are either identical or highly conserved to the FACS signature motif. Eighteen site-directed mutations within the fatty acyl-CoA synthetase structural gene (fadD) corresponding to this motif were constructed to evaluate the contribution of this region of the enzyme to catalytic activity. Three distinct classes of mutations were identified on the basis of growth characteristics on fatty acids, enzymatic activities using cell extracts, and studies using purified wild-type and mutant forms of the enzyme: 1) those that resulted in either wild-type or nearly wild-type fatty acyl-CoA synthetase activity profiles; 2) those that had little or no enzyme activity; and 3) those that resulted in lowering and altering fatty acid chain length specificity. Among the 18 mutants characterized, 7 fall in the third class. We propose that the FACS signature motif is essential for catalytic activity and functions in part to promote fatty acid chain length specificity and thus may compose part of the fatty acid binding site within the enzyme.",
author = "Black, {Paul N.} and Qing Zhang and Weimar, {James D.} and DiRusso, {Concetta C.}",
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T1 - Mutational analysis of a fatty acyl-coenzyme a synthetase signature motif identifies seven amino acid residues that modulate fatty acid substrate specificity

AU - Black, Paul N.

AU - Zhang, Qing

AU - Weimar, James D.

AU - DiRusso, Concetta C.

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N2 - Fatty acyl-CoA synthetase (fatty acid:CoA ligase, AMP-forming; EC 6.2.1.3) catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of pyrophosphate. In Escherichia coli this enzyme plays a pivotal role in the uptake of long chain fatty acids (C12- C18) and in the regulation of the global transcriptional regulator FadR. The E. coli fatty acyl-CoA synthetase has remarkable amino acid similarities and identities to the family of both prokaryotic and eukaryotic fatty acyl-CoA synthetases, indicating a common ancestry. Most notable in this regard is a 25-amino acid consensus sequence, DGWLHTGDIGXWX-PXGXLKIIDRKK, common to all fatty acyl-CoA synthetases for which sequence information is available. Within this consensus are 8 invariant and 13 highly conserved amino acid residues in the 12 fatty acyl-CoA synthetases compared. We propose that this sequence represents the fatty acyl-CoA synthetase signature motif (FACS signature motif). This region of fatty acyl-CoA synthetase from E. coli, 431NGWLHTGDIAVMDEEGFL-RIVDRKK 455, contains 17 amino acid residues that are either identical or highly conserved to the FACS signature motif. Eighteen site-directed mutations within the fatty acyl-CoA synthetase structural gene (fadD) corresponding to this motif were constructed to evaluate the contribution of this region of the enzyme to catalytic activity. Three distinct classes of mutations were identified on the basis of growth characteristics on fatty acids, enzymatic activities using cell extracts, and studies using purified wild-type and mutant forms of the enzyme: 1) those that resulted in either wild-type or nearly wild-type fatty acyl-CoA synthetase activity profiles; 2) those that had little or no enzyme activity; and 3) those that resulted in lowering and altering fatty acid chain length specificity. Among the 18 mutants characterized, 7 fall in the third class. We propose that the FACS signature motif is essential for catalytic activity and functions in part to promote fatty acid chain length specificity and thus may compose part of the fatty acid binding site within the enzyme.

AB - Fatty acyl-CoA synthetase (fatty acid:CoA ligase, AMP-forming; EC 6.2.1.3) catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of pyrophosphate. In Escherichia coli this enzyme plays a pivotal role in the uptake of long chain fatty acids (C12- C18) and in the regulation of the global transcriptional regulator FadR. The E. coli fatty acyl-CoA synthetase has remarkable amino acid similarities and identities to the family of both prokaryotic and eukaryotic fatty acyl-CoA synthetases, indicating a common ancestry. Most notable in this regard is a 25-amino acid consensus sequence, DGWLHTGDIGXWX-PXGXLKIIDRKK, common to all fatty acyl-CoA synthetases for which sequence information is available. Within this consensus are 8 invariant and 13 highly conserved amino acid residues in the 12 fatty acyl-CoA synthetases compared. We propose that this sequence represents the fatty acyl-CoA synthetase signature motif (FACS signature motif). This region of fatty acyl-CoA synthetase from E. coli, 431NGWLHTGDIAVMDEEGFL-RIVDRKK 455, contains 17 amino acid residues that are either identical or highly conserved to the FACS signature motif. Eighteen site-directed mutations within the fatty acyl-CoA synthetase structural gene (fadD) corresponding to this motif were constructed to evaluate the contribution of this region of the enzyme to catalytic activity. Three distinct classes of mutations were identified on the basis of growth characteristics on fatty acids, enzymatic activities using cell extracts, and studies using purified wild-type and mutant forms of the enzyme: 1) those that resulted in either wild-type or nearly wild-type fatty acyl-CoA synthetase activity profiles; 2) those that had little or no enzyme activity; and 3) those that resulted in lowering and altering fatty acid chain length specificity. Among the 18 mutants characterized, 7 fall in the third class. We propose that the FACS signature motif is essential for catalytic activity and functions in part to promote fatty acid chain length specificity and thus may compose part of the fatty acid binding site within the enzyme.

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