Fatty acyl-CoA binding domain of the transcription factor FadR: Characterization by deletion, affinity labeling, and isothermal titration calorimetry

Concetta C. DiRusso, Vadim Tsvetnitsky, Peter Højrup, Jens Knudsen

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

The Escherichia coli transcription factor FadR regulates genes required for fatty acid biosynthesis and degradation in an opposing manner. It is acting as an activator of biosynthetic genes and a repressor of degradative genes. The DNA binding of FadR to regions within the promoters of responsive genes and operons is inhibited by long chain acyl-CoA thioesters but not free fatty acids or coenzyme A. The acyl-CoA binding domain of FadR was localized by affinity labeling of the full-length protein and an amino-terminal deletion derivative, FadRΔ1-167, with a palmitoyl-CoA analogue, 9-p- azidophenoxy[9-3H]nonanoic acid. CoA ester. Analysis of labeled peptides generated by tryptic digestion of the affinity-labeled proteins identified one peptide common to both the full-length protein and the deletion derivative. The amino-terminal sequence of the labeled peptide was SLALGFYHK, which corresponds to amino acids 187-195 in FadR. Isothermal titration calorimetry was used to estimate affinity of the wild-type full-length FadR, a His-tagged derivative, and FadRΔ1-167 for acyl-CoA. The binding was characterized by a large negative ΔH0 -16 to -20 kcal mol-1. No binding was detected for the medium chain ligand C8-CoA. Full-length wild-type FadR and His6-FadR bound oleoyl-CoA and myristoyl-CoA with similar affinities, K(d) of 45 and 63 nM and 68 and 59 nM, respectively. The K(d) for palmitoyl- CoA binding was about 5-fold higher despite the fact that palmitoyl-CoA is 50-fold more efficient in inhibiting FadR binding to DNA than myristoyl-CoA. The results indicate that both acyl-CoA chain length and the presence of double bonds in the acyl chain affect FadR ligand binding.

Original languageEnglish (US)
Pages (from-to)33652-33659
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number50
DOIs
StatePublished - Dec 11 1998

Fingerprint

Acyl Coenzyme A
Calorimetry
Palmitoyl Coenzyme A
Titration
Labeling
Transcription Factors
Coenzyme A
Genes
Derivatives
Peptides
Ligands
Proteins
DNA
Biosynthesis
Operon
Chain length
Nonesterified Fatty Acids
Genetic Promoter Regions
Escherichia coli
Digestion

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Fatty acyl-CoA binding domain of the transcription factor FadR : Characterization by deletion, affinity labeling, and isothermal titration calorimetry. / DiRusso, Concetta C.; Tsvetnitsky, Vadim; Højrup, Peter; Knudsen, Jens.

In: Journal of Biological Chemistry, Vol. 273, No. 50, 11.12.1998, p. 33652-33659.

Research output: Contribution to journalArticle

@article{25d31f803d8f4a72b88014d43ca29346,
title = "Fatty acyl-CoA binding domain of the transcription factor FadR: Characterization by deletion, affinity labeling, and isothermal titration calorimetry",
abstract = "The Escherichia coli transcription factor FadR regulates genes required for fatty acid biosynthesis and degradation in an opposing manner. It is acting as an activator of biosynthetic genes and a repressor of degradative genes. The DNA binding of FadR to regions within the promoters of responsive genes and operons is inhibited by long chain acyl-CoA thioesters but not free fatty acids or coenzyme A. The acyl-CoA binding domain of FadR was localized by affinity labeling of the full-length protein and an amino-terminal deletion derivative, FadRΔ1-167, with a palmitoyl-CoA analogue, 9-p- azidophenoxy[9-3H]nonanoic acid. CoA ester. Analysis of labeled peptides generated by tryptic digestion of the affinity-labeled proteins identified one peptide common to both the full-length protein and the deletion derivative. The amino-terminal sequence of the labeled peptide was SLALGFYHK, which corresponds to amino acids 187-195 in FadR. Isothermal titration calorimetry was used to estimate affinity of the wild-type full-length FadR, a His-tagged derivative, and FadRΔ1-167 for acyl-CoA. The binding was characterized by a large negative ΔH0 -16 to -20 kcal mol-1. No binding was detected for the medium chain ligand C8-CoA. Full-length wild-type FadR and His6-FadR bound oleoyl-CoA and myristoyl-CoA with similar affinities, K(d) of 45 and 63 nM and 68 and 59 nM, respectively. The K(d) for palmitoyl- CoA binding was about 5-fold higher despite the fact that palmitoyl-CoA is 50-fold more efficient in inhibiting FadR binding to DNA than myristoyl-CoA. The results indicate that both acyl-CoA chain length and the presence of double bonds in the acyl chain affect FadR ligand binding.",
author = "DiRusso, {Concetta C.} and Vadim Tsvetnitsky and Peter H{\o}jrup and Jens Knudsen",
year = "1998",
month = "12",
day = "11",
doi = "10.1074/jbc.273.50.33652",
language = "English (US)",
volume = "273",
pages = "33652--33659",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "50",

}

TY - JOUR

T1 - Fatty acyl-CoA binding domain of the transcription factor FadR

T2 - Characterization by deletion, affinity labeling, and isothermal titration calorimetry

AU - DiRusso, Concetta C.

AU - Tsvetnitsky, Vadim

AU - Højrup, Peter

AU - Knudsen, Jens

PY - 1998/12/11

Y1 - 1998/12/11

N2 - The Escherichia coli transcription factor FadR regulates genes required for fatty acid biosynthesis and degradation in an opposing manner. It is acting as an activator of biosynthetic genes and a repressor of degradative genes. The DNA binding of FadR to regions within the promoters of responsive genes and operons is inhibited by long chain acyl-CoA thioesters but not free fatty acids or coenzyme A. The acyl-CoA binding domain of FadR was localized by affinity labeling of the full-length protein and an amino-terminal deletion derivative, FadRΔ1-167, with a palmitoyl-CoA analogue, 9-p- azidophenoxy[9-3H]nonanoic acid. CoA ester. Analysis of labeled peptides generated by tryptic digestion of the affinity-labeled proteins identified one peptide common to both the full-length protein and the deletion derivative. The amino-terminal sequence of the labeled peptide was SLALGFYHK, which corresponds to amino acids 187-195 in FadR. Isothermal titration calorimetry was used to estimate affinity of the wild-type full-length FadR, a His-tagged derivative, and FadRΔ1-167 for acyl-CoA. The binding was characterized by a large negative ΔH0 -16 to -20 kcal mol-1. No binding was detected for the medium chain ligand C8-CoA. Full-length wild-type FadR and His6-FadR bound oleoyl-CoA and myristoyl-CoA with similar affinities, K(d) of 45 and 63 nM and 68 and 59 nM, respectively. The K(d) for palmitoyl- CoA binding was about 5-fold higher despite the fact that palmitoyl-CoA is 50-fold more efficient in inhibiting FadR binding to DNA than myristoyl-CoA. The results indicate that both acyl-CoA chain length and the presence of double bonds in the acyl chain affect FadR ligand binding.

AB - The Escherichia coli transcription factor FadR regulates genes required for fatty acid biosynthesis and degradation in an opposing manner. It is acting as an activator of biosynthetic genes and a repressor of degradative genes. The DNA binding of FadR to regions within the promoters of responsive genes and operons is inhibited by long chain acyl-CoA thioesters but not free fatty acids or coenzyme A. The acyl-CoA binding domain of FadR was localized by affinity labeling of the full-length protein and an amino-terminal deletion derivative, FadRΔ1-167, with a palmitoyl-CoA analogue, 9-p- azidophenoxy[9-3H]nonanoic acid. CoA ester. Analysis of labeled peptides generated by tryptic digestion of the affinity-labeled proteins identified one peptide common to both the full-length protein and the deletion derivative. The amino-terminal sequence of the labeled peptide was SLALGFYHK, which corresponds to amino acids 187-195 in FadR. Isothermal titration calorimetry was used to estimate affinity of the wild-type full-length FadR, a His-tagged derivative, and FadRΔ1-167 for acyl-CoA. The binding was characterized by a large negative ΔH0 -16 to -20 kcal mol-1. No binding was detected for the medium chain ligand C8-CoA. Full-length wild-type FadR and His6-FadR bound oleoyl-CoA and myristoyl-CoA with similar affinities, K(d) of 45 and 63 nM and 68 and 59 nM, respectively. The K(d) for palmitoyl- CoA binding was about 5-fold higher despite the fact that palmitoyl-CoA is 50-fold more efficient in inhibiting FadR binding to DNA than myristoyl-CoA. The results indicate that both acyl-CoA chain length and the presence of double bonds in the acyl chain affect FadR ligand binding.

UR - http://www.scopus.com/inward/record.url?scp=0032509447&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032509447&partnerID=8YFLogxK

U2 - 10.1074/jbc.273.50.33652

DO - 10.1074/jbc.273.50.33652

M3 - Article

C2 - 9837950

AN - SCOPUS:0032509447

VL - 273

SP - 33652

EP - 33659

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 50

ER -