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 language | English (US) |
|---|---|
| Pages (from-to) | 33652-33659 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 273 |
| Issue number | 50 |
| DOIs | |
| State | Published - Dec 11 1998 |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology
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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 journal › Article
}
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.
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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 -