Analysis of acyl coenzyme A binding to the transcription factor FadR and identification of amino acid residues in the carboxyl terminus required for ligand binding

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Abstract

The Escherichia coli FadR protein regulates the transcription of many unlinked genes and operons encoding proteins required for fatty acid synthesis and degradation. Previously, we demonstrated that the ability of purified FadR to bind DNA in vitro is inhibited by long chain acyl coenzyme A esters (DiRusso, D. D., Heimert, T. L., and Metzger, A. K. (1992) J. Biol. Chem. 267, 8685-8691). In the present work, we show that FadR binds acyl-CoA directly. Ligand binding resulted in a shift in the apparent pI of FadR from 6.9 to 6.2 and in a marked decrease in intrinsic fluorescence. The K(m) for FadR binding of oleoyl coenzyme A was determined to be 12.1 nM using the fluorescence quenching assay. The binding site for acyl-CoA was identified by selection of non-inducible mutations in the FadR gene. One altered protein carrying the change Ser219 to Asn (S219N) was purified and shown to have a reduced affinity for oleoyl coenzyme A as evidenced by a K(m) of 257 nM. S219N retained the ability to bind DNA and to repress or activate transcription. Alanine substitution of amino acid residues 215 through 230 identified Gly216 and Trp223 as also required specifically for induction. This region of FadR shares amino acid identities and similarities with the coenzyme A-binding site of Clostridium thermoaceticum CO dehydrogenase/acetyl-coenzyme A synthase. Due to the alteration in binding affinity of the purified S219N protein, the non-inducible phenotype of several proteins carrying alanine substitutions and similarities to CO dehydrogenase/acetyl-coenzyme A synthase we propose this region of FadR forms part of the acyl-CoA-binding domain.

Original languageEnglish (US)
Pages (from-to)1092-1097
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number3
DOIs
StatePublished - Jan 1 1995

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Acyl Coenzyme A
carbon monoxide dehydrogenase
Transcription Factors
Ligands
Amino Acids
Acetyl Coenzyme A
Alanine
Transcription
Proteins
Fluorescence
Binding Sites
Substitution reactions
Genes
Clostridium
Escherichia coli Proteins
DNA
Amino Acid Substitution
Coenzyme A
Operon
Esters

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Analysis of acyl coenzyme A binding to the transcription factor FadR and identification of amino acid residues in the carboxyl terminus required for ligand binding",
abstract = "The Escherichia coli FadR protein regulates the transcription of many unlinked genes and operons encoding proteins required for fatty acid synthesis and degradation. Previously, we demonstrated that the ability of purified FadR to bind DNA in vitro is inhibited by long chain acyl coenzyme A esters (DiRusso, D. D., Heimert, T. L., and Metzger, A. K. (1992) J. Biol. Chem. 267, 8685-8691). In the present work, we show that FadR binds acyl-CoA directly. Ligand binding resulted in a shift in the apparent pI of FadR from 6.9 to 6.2 and in a marked decrease in intrinsic fluorescence. The K(m) for FadR binding of oleoyl coenzyme A was determined to be 12.1 nM using the fluorescence quenching assay. The binding site for acyl-CoA was identified by selection of non-inducible mutations in the FadR gene. One altered protein carrying the change Ser219 to Asn (S219N) was purified and shown to have a reduced affinity for oleoyl coenzyme A as evidenced by a K(m) of 257 nM. S219N retained the ability to bind DNA and to repress or activate transcription. Alanine substitution of amino acid residues 215 through 230 identified Gly216 and Trp223 as also required specifically for induction. This region of FadR shares amino acid identities and similarities with the coenzyme A-binding site of Clostridium thermoaceticum CO dehydrogenase/acetyl-coenzyme A synthase. Due to the alteration in binding affinity of the purified S219N protein, the non-inducible phenotype of several proteins carrying alanine substitutions and similarities to CO dehydrogenase/acetyl-coenzyme A synthase we propose this region of FadR forms part of the acyl-CoA-binding domain.",
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T1 - Analysis of acyl coenzyme A binding to the transcription factor FadR and identification of amino acid residues in the carboxyl terminus required for ligand binding

AU - Raman, N.

AU - DiRusso, Concetta C

PY - 1995/1/1

Y1 - 1995/1/1

N2 - The Escherichia coli FadR protein regulates the transcription of many unlinked genes and operons encoding proteins required for fatty acid synthesis and degradation. Previously, we demonstrated that the ability of purified FadR to bind DNA in vitro is inhibited by long chain acyl coenzyme A esters (DiRusso, D. D., Heimert, T. L., and Metzger, A. K. (1992) J. Biol. Chem. 267, 8685-8691). In the present work, we show that FadR binds acyl-CoA directly. Ligand binding resulted in a shift in the apparent pI of FadR from 6.9 to 6.2 and in a marked decrease in intrinsic fluorescence. The K(m) for FadR binding of oleoyl coenzyme A was determined to be 12.1 nM using the fluorescence quenching assay. The binding site for acyl-CoA was identified by selection of non-inducible mutations in the FadR gene. One altered protein carrying the change Ser219 to Asn (S219N) was purified and shown to have a reduced affinity for oleoyl coenzyme A as evidenced by a K(m) of 257 nM. S219N retained the ability to bind DNA and to repress or activate transcription. Alanine substitution of amino acid residues 215 through 230 identified Gly216 and Trp223 as also required specifically for induction. This region of FadR shares amino acid identities and similarities with the coenzyme A-binding site of Clostridium thermoaceticum CO dehydrogenase/acetyl-coenzyme A synthase. Due to the alteration in binding affinity of the purified S219N protein, the non-inducible phenotype of several proteins carrying alanine substitutions and similarities to CO dehydrogenase/acetyl-coenzyme A synthase we propose this region of FadR forms part of the acyl-CoA-binding domain.

AB - The Escherichia coli FadR protein regulates the transcription of many unlinked genes and operons encoding proteins required for fatty acid synthesis and degradation. Previously, we demonstrated that the ability of purified FadR to bind DNA in vitro is inhibited by long chain acyl coenzyme A esters (DiRusso, D. D., Heimert, T. L., and Metzger, A. K. (1992) J. Biol. Chem. 267, 8685-8691). In the present work, we show that FadR binds acyl-CoA directly. Ligand binding resulted in a shift in the apparent pI of FadR from 6.9 to 6.2 and in a marked decrease in intrinsic fluorescence. The K(m) for FadR binding of oleoyl coenzyme A was determined to be 12.1 nM using the fluorescence quenching assay. The binding site for acyl-CoA was identified by selection of non-inducible mutations in the FadR gene. One altered protein carrying the change Ser219 to Asn (S219N) was purified and shown to have a reduced affinity for oleoyl coenzyme A as evidenced by a K(m) of 257 nM. S219N retained the ability to bind DNA and to repress or activate transcription. Alanine substitution of amino acid residues 215 through 230 identified Gly216 and Trp223 as also required specifically for induction. This region of FadR shares amino acid identities and similarities with the coenzyme A-binding site of Clostridium thermoaceticum CO dehydrogenase/acetyl-coenzyme A synthase. Due to the alteration in binding affinity of the purified S219N protein, the non-inducible phenotype of several proteins carrying alanine substitutions and similarities to CO dehydrogenase/acetyl-coenzyme A synthase we propose this region of FadR forms part of the acyl-CoA-binding domain.

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