©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 (*)

(Received for publication, August 10, 1994; and in revised form, October 21, 1994)

Narayan Raman Concetta C. DiRusso (§)

From the Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 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 noninducible mutations in the FadR gene. One altered protein carrying the change Ser to Asn (S219N) was purified and shown to have a reduced affinity for oleoyl coenzyme A as evidenced by a K 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 Gly and Trp 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.


INTRODUCTION

The Escherichia coli fadR gene product, FadR, plays a critical role in the regulation many genes required for fatty acid metabolism. Specifically FadR is a repressor of transcription of genes which are required for fatty acid transport and beta-oxidation including fadL, fadD, fadE, fadBA, and fadH. FadR is an activator of transcription of the fabA gene which is required for unsaturated fatty acid biosynthesis (reviewed in (1) ). Induction of FadR-dependent expression of the fatty acid degradative (fad) genes occurs upon growth of E. coli in medium containing long chain fatty acids (C(14)-C(18)). The same growth conditions result in an decrease in fabA expression.

It has been demonstrated in vitro that FadR exerts its effect at the level of transcription by directly binding to DNA to repress the expression of the fad genes and to activate the expression of fabA(2, 3) . Six FadR binding sites have been identified by footprinting analyses in the promoter regions of four FadR-responsive genes giving a consensus sequence which is 5`-AGCTGGTCCGAYNTGTT-3`(1, 2, 3, 4) . FadR-specific DNA binding in vitro is prevented by the addition of long chain acyl-CoA esters (2, 5) . Medium chain acyl-CoA esters or free fatty acids do not prevent DNA binding(2) . Therefore, we concluded that long chain acyl-CoA esters are the effector molecules that regulate fatty acid metabolism at the level of FadR-dependent transcription in E. coli(2) . However, this conclusion was based on an indirect measurement of FadR-acyl-CoA interaction using protein-DNA electrophoretic mobility shift assays. The present study had two goals, to identify mutations in fadR which specifically altered effector binding and to demonstrate that long chain acyl-CoA esters bind directly to FadR and thereby mediate induction.

The mutational analyses reported here selected fadR alleles that were non-inducible by long chain fatty acids but which maintained the ability to repress or activate transcription (termed super-repressors). Such mutants were expected to identify amino acid residues within FadR specifically required for induction. The experiments reported in this study show that 5 amino acid residues Gly, Glu, Ser, Trp, and Lys near the carboxyl terminus of FadR were required for maximal levels of induction by long chain fatty acids.

Additionally, we measured FadR-acyl-CoA binding directly by using isoeletric focusing and by quenching of intrinsic fluorescence. Our results demonstrated that purified FadR binds long chain acyl-CoA with high affinity and that the altered phenotype of the fadR allele S219N was correlated with a reduced affinity for long chain acyl-CoA in vitro. Due to amino acid similarities and identities between the region of FadR identified by this mutational analysis and the CoA-binding site of the CO dehydrogenase/acetyl-coenzyme A synthase (CODH) (^1)of Clostridium thermoaceticum identified by biochemical studies (6, 7) we propose that the carboxyl-terminal region of FadR including particularly amino acids 216-228 binds the CoA moiety of the long chain acyl-CoA ligand.


EXPERIMENTAL PROCEDURES

Strains and Growth Conditions

E. coli strains used in this study were: LS1085, F fadR, thi1, leuB6, lacY1, tonA21, supE44, , for characterization of fadR phenotype on minimal media containing fatty acids and for plasmid propagation(2) ; JM109, endA1, recA1, gyrA96, thi, hsdR17 (r(k)-, m(k)+), relA1, supE44, Delta(lac-proAB), - F` traD36, proAB, lacI^qZDeltaM15, for propagation of pSELECT-1 derived phagemids(8) ; LS1346, fadR, araD139, Delta(lacIOPZYA)U169, rpsL, thiA/fadB-lacZ was used to measure in vivo beta-oxidation and to assess FadR-dependent repression of transcription from fadB(3) ; LS1348, fadR, araD139, Delta(lacIOPZYA)U169, rpsL, thiA/fabA-lacZ to assess FadR-dependent activation of fabA(3) ; and BL21(DE3)/pLysS F- recA, r(k)-, m(k)+Rif^r, for controlled expression of FadR(9) . For growth of cells for biochemical assays, tryptone broth (TB, which contained 10 g of Difco bacto tryptone and 5 g of sodium chloride/liter) was used. When necessary to maintain plasmids antibiotics were added at a final concentration of 100 µg/ml ampicillin, 40 µg/ml kanamycin, or 12.5 µg/ml chloramphenicol. The minimal medium used was medium E(10) . Fatty acids were provided at 5 mM in 0.5% Brij 58. Liquid cultures were grown at 37 °C with shaking in a New Brunswick gyratory incubator. Bacterial growth was monitored in a Klett Summerson Colorimeter equipped with a blue filter.

Plasmid Constructions

The plasmid pCD126 was constructed by cloning an 800-base pair HindIII-TaqI fragment carrying wild-type fadR into HindIII-ClaI restricted pACYC177(11) . For overexpression, S219N encoded within pRW22 was subcloned to pT7-5 by excision of a HindIII-XhoI fragment containing fadR, addition of an oligonucleotide linker to convert the XhoI site to a HindIII site, and insertion of the HindIII fragment into HindIII digested pT7-5 to generate pCD22.

Hydroxylamine Mutagenesis

pCD126 DNA (50 µg) was incubated in 100 µl of 0.8 M hydroxylamine, 1 mM EDTA, 50 mM sodium phosphate, pH 7.6, at 37 °C. Aliquots were removed at 24, 36, 48, and 96 h, ethanol precipitated, resuspended in 25 µl of 10 mM Tris, 1 mM EDTA, pH 7.5, and dialyzed against 1 liter of the same buffer. The mutagenized DNA was transformed into LS1085 and plated onto LB plates containing ampicillin, and then the transformed colonies were tested for growth on minimal plates containing oleate or decanoate. Two-thousand colonies were screened and three were identified as putative non-inducible super-repressors by an inability to grow on minimal media containing oleate or decanoate (ole dec) and by non-inducible levels of in vivo beta-oxidation as compared with the same strain carrying pACYC177 or pCD126.

Site-directed Mutagenesis

Specific amino acid substitutions in fadR were constructed using the Altered Sites System (Promega). A 4-kilobase pair HinDIII fragment encoding FadR was subcloned from pCD103 (previously called pACfadR3(12) ) to pSELECT-1 to generate pCD152. fadR-specific oligonucleotides (24-27 bases in length) were synthesized carrying an alanine codon substitution in place of the amino acid of interest. Mutagenesis was carried out according to the manufacturer. Mutations were identified by sequencing. For analysis of the mutant proteins, a HinDIII-BamHI fragment was removed from the pCD152 derivative and subcloned to pACYC177.

Preparation of Mutant Proteins and DNA Binding Assays

Wild-type and S219N FadR proteins were overexpressed and purified as previously detailed(2) . Affinity of wild-type or mutant FadR for O(B) was assessed using the 377-base pair DNA fragment carrying the fadB promoter obtained from pCD154 after treatment with HinDIII and EcoRI. The DNA-protein gel shift assay was performed as previously detailed(2) .

Biochemical Procedures

The level of beta-oxidation in whole cells was measured as the release of [^14C]CO(2) from 1-[^14C]oleate after growth in TB using 5 mM oleate as described in Simons et al.(13) .

For the assay of beta-galactosidase activity in strains carrying fadB-lacZ or fabA-lacZ, cells were grown in 10 ml of TB to mid-log phase, rinsed twice with Z buffer, resuspended to 5 times 10^8 cells/ml, and lysed by sonication. beta-Galactosidase activity was assayed immediately using o-nitrophenyl-beta-D-galactopyranoside as substrate as described by Miller(10) . Specific activities were calculated using a value of 1.25 µg of protein/Klett unit.

DNA Sequencing

The fadR allele encoded within pRW22 was sequenced in its entirety across both DNA strands by the dideoxy chain-terminating method using 10 fadR-specific primers, each 17 nucleotides in length. The double-stranded DNA (2-5 µg) was denatured by incubation for 15 min in 20 µl of 0.2 N NaOH and 0.5 mM EDTA. The solution was neutralized by the addition of 5 µl of 5 M sodium acetate, pH 4.8, and DNA was precipitated by the addition of 2 volumes of ethanol. The DNA was centrifuged, rinsed, dried, and sequenced using Sequenase and a reagent kit purchased from United States Biochemical Corp. (Cleveland OH). Mutants generated using the Altered Site system were sequenced using a fadR primer which read across a 250-base pair region which included the site of the mutation which had been introduced.

Isoelectric Focusing

The isoelectric point of native FadR or FadR complexed with acyl-CoA was determined by electrophoresis at 10 °C on pH 3-10 isoelectric focusing gels (Isolab, Inc.) using a Hoeffer HE950 flatbed apparatus. Five µl of 4.5 µM FadR in 20 mM sodium phosphate, pH 7.5, 250 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol was applied one-quarter of the distance of the gel from the cathode. The sample was electrophoresed for 10 min at 1 watt constant power, the sample template was removed, and electrophoresis continued for 1 h at 10 watts constant power. The gels were fixed in glutaraldehyde, dried, and the protein detected by staining with silver nitrate as recommended by the manufacturer.

Fluorescence Spectroscopy

Fluorescence studies were performed with an Aminco Bowman Series 2 luminescence spectrometer according to the principles given by Ward(15) . Emission spectra from 290 to 400 nm were recorded at room temperature in a stirred cuvette containing purified FadR (0.12 µM in 20 mM Tris, pH 7.5) with excitation at 285 nm, a band pass of 8 nm, and step size of 1. There were no corrections made for inner filter effect. To estimate FadR-acyl-CoA binding, we measured the relative decrease of intrinsic fluorescence of purified FadR upon addition of long chain acyl-CoA compounds by titrating acyl-CoA and scanning the protein at approximately 10-min intervals. The quenching of fluorescence occurred rapidly and was stable for at least 1 h as determined using multiple scans of the same sample.

Enzymes and Radiochemicals

Restriction enzymes, T4 DNA ligase, T4 DNA Polymerase, Klenow DNA polymerase, and Sequenase V.2 were purchased from U. S. Biochemicals Inc. [alpha-P]dATP and 1-[^14C]oleate (57 Ci/mmol) were purchased from DuPont NEN.


RESULTS

Mutagenesis and Identification of a FadR Superrepressor

Plasmid pCD126 encoding wild-type FadR was treated with hydroxylamine in vitro and then used to transform the fadR strain LS1085. Transformants were selected on rich media containing ampicillin and were subsequently tested for the ability to grow on minimal media containing oleate (C) or decanoate (C) as sole carbon and energy source. Approximately 1.5% of the colonies were able to grow on both oleate and decanoate which is the usual phenotype of fadR strains. Most of the transformants appeared to be wild-type since they were able to grow on media containing oleate but not decanoate (fadR). This is because long chain fatty acids but not medium chain fatty acids induce FadR and allow expression of the fad genes which encode proteins required for fatty acid transport and degradation by beta-oxidation. Three isolates were unable to grow on media containing either fatty acid (fadR^S). Plasmids isolated from these strains retransformed the ole dec phenotype. However, two gave mixed phenotypes upon retransformation and were not stable to propagation. Only one of these plasmids identified after hydroxylamine mutagenesis, pRW22, has proven stable to propagation and allowed subsequent analysis. It is possible that the production of the mutant FadR proteins encoded by the unstable isolates was toxic.

To assess inducibility, plasmid DNA was transformed into strain LS1346 (fadR) and in vivo rates of beta-oxidation were measured after growth under inducing (TBO, tryptone broth containing 5 mM oleate) or non-inducing conditions (TB, tryptone broth). These results, presented in Table 1, demonstrated that FadR encoded within pRW22 resulted in very low levels of beta-oxidation, which were induced 2-3-fold as compared with 18-fold for wild-type FadR. These low levels of in vivo beta-oxidation were correlated with the inability of E. coli cells harboring pRW22 to use long chain fatty acids as a carbon and energy source. The fadR allele encoded within pRW22 was sequenced in its entirety across both strands. A single amino acid substitution of serine at position 219 for asparagine (S219N) resulted from a codon change of AGT to AAT.



Transcriptional Regulation of fadB and fabA by S219N

FadR acts as both a repressor of genes encoding proteins required for growth on fatty acids and as an activator of at least one gene required for unsaturated fatty acid biosynthesis, therefore we tested the ability of S219N to repress or activate transcription from a lacZ reporter. beta-Galactosidase activities were measured in LS1346 which is fadR fadB-lacZ and LS1348 which is fadR fabA-lacZ(3) (Table 1). Basal and induced levels of beta-galactosidase of strain LS1346 transformed with the vector pACYC177 were 8-fold higher than those obtained for the same strain transformed with wild-type FadR grown under non-inducing conditions. LS1346 transformed with pCD126 encoding wild-type FadR and grown under inducing conditions had levels four times the same strain grown under non-inducing conditions, showing the usual pattern of induction for wild-type fadR. In contrast, transformants of pRW22 encoding S219N had very low levels of beta-galactosidase whether grown under inducing or non-inducing conditions.

beta-Galactosidase activities for LS1348 (fadR fabA-lacZ) transformed with wild-type or S219N were 14- and 10-fold higher, respectively, than control cells. Addition of oleate to the growth medium caused a 50% reduction in activity for LS1348 transformed with pCD126 (wild-type FadR) as compared with an 11% reduction for cells transformed with pRW22 (S219N). Therefore, S219N retained the ability to bind DNA and to repress or activate transcription. However, the change in activity with regard to repression or activation in response to long chain fatty acids in the growth medium was reduced for S219N as compared with wild-type FadR.

Purification of S219N and Analysis of DNA Binding

There are at least three mechanisms through which S219N could result in the non-inducible phenotype: (i) an increased ability to bind DNA; (ii) a decreased ability to bind acyl-CoA; or (iii) an inability of the altered protein to undergo the allosteric transition which results in a conformation unfavorable for DNA binding. To distinguish between these possibilities, we overexpressed and purified S219N using the T7 expression system as previously detailed for wild-type FadR(2) . The purified protein was tested for the ability to bind DNA containing the FadR-binding site within fadB (O(B)) using the DNA-protein electrophoretic mobility shift assay (gel shift) (detailed in (2) ). The affinity of wild-type FadR for DNA containing O(B) was 3 ± 1 times 10 and the affinity of S219N was 5 ± 2 times 10 (Fig. 1). Therefore, increased DNA binding did not appear to account for the non-inducible phenotype.


Figure 1: DNA binding affinity of wild-type and S219N FadR proteins. Gel shift experiments were performed using purified FadR and 1 times 10 DNA containing O(B) as described under ``Experimental Procedures.'' The % Bound indicates the amount of DNA migrating as the slow form relative to the total amount of DNA (Bound plus Unbound fractions) in a gel shift experiment. The concentration of FadR was as indicated.



We also tested whether acyl-CoA would prevent DNA binding. In these experiments 5 nM FadR was mixed with varying concentrations of C-CoA and 1 times 10 [P]O(B) DNA. DNA bound by FadR was separated and detected from unbound DNA on standard 5% native gels (Fig. 2). These experiments showed that the apparent K(i) for inhibition of DNA binding by C-CoA was 10-fold higher for S219N than for wild-type FadR. A similar change in apparent K(i) was determined for C-CoA.


Figure 2: Inhibition of DNA binding by C-CoA. Gel shift analysis was performed with DNA containing O(B) as described under ``Experimental Procedures.'' Lane R indicates DNA alone; lane 0 indicates 5 nM wild-type or S219N FadR as indicated. Lanes 1, 2, 5, 10, 50, and 100 indicate the nanomolar concentration of C-CoA added to a similar reaction containing 5 nM FadR.



Analysis of Acyl-CoA Binding

While there was a change in apparent K(i) of FadR-specific DNA binding by long chain acyl-CoA compounds assayed using gel shift assays, this method was an indirect measure of FadR-acyl-CoA interaction. To directly evaluate binding, two methods were employed, isoelectric focusing and quenching of intrinsic fluorescence.

A change in the apparent isoelectric point upon binding the substrate myristoyl-CoA was previously demonstrated for N-myristoyl-CoA-acyl-transferase(16) . It was suggested that the apparent change in isoelectric point occurs as a result of surface charge redistribution upon ligand binding to the native protein. The pI of FadR was determined to be 6.9 by displaying the purified protein on isoelectric focusing gels with a pH range of 3 to 10. Upon addition of long chain acyl-CoA the apparent pI shifts to 6.2 (Fig. 3). The shift in pI did not occur upon addition of C-CoA, CoA, or long chain fatty acid (Fig. 3). This follows the same pattern of induction of beta-oxidation and fad gene activity observed in in vivo studies and the pattern observed for inhibition of DNA binding using the gel shift assay whereby induction was shown to be specific for long chain fatty acids and DNA binding was specifically prevented by the long chain acyl-coenzyme derivatives. When purified S219N was mixed with acyl-CoA under the same conditions a partial shift of the protein was noted only at the highest concentrations tested (10-fold molar excess of ligand over protein) (Fig. 3).


Figure 3: Alteration of the isoelectric point of FadR by acyl-CoA. Lanes 1-8 contain wild type FadR (4.5 µM). Lane 1, no addition; lane 2, 50 µM C-CoA; lane 3, 25 µM C-CoA; lane 4, 10 µM C-CoA; lane 5, 5 µM C-CoA; lane 6, 50 µM CoA; lane 7, 50 µM C-CoA; lane 8, 50 µM C. Lanes 9 and 10 contain S219N (4.5 µM). Lane 9, no additions, and lane 10 contains 50 µM C-CoA. The pI of standard proteins are given on the right. The open arrow indicates the position of FadR alone and the closed arrow indicates the position of FadR complexed with acyl-CoA.



In order to estimate the affinity constant of FadR-acyl-CoA binding, we measured quenching of intrinsic fluorescence of purified protein upon addition of long chain acyl-CoA. Purified FadR (0.12 µM) was excited at 285 nm and an emission spectrum was recorded from 290 to 400 nm. There was no discernable difference in the spectra observed for wild-type or S219N proteins without added ligand. Upon addition of C-CoA there was a marked decrease in observed fluorescence for wild-type FadR (Fig. 4A). When C-CoA was added to a solution containing FadR there was a small decrease in intrinsic fluorescence (approximately 10% at 1 µM C-CoA) which could not be used to determine an affinity constant for either the wild-type or mutant proteins. The decrease in intrinsic fluorescence for C-CoA was used to quantitate FadR-acyl-CoA binding affinity by titration of FadR with acyl-CoA. The data were analyzed using the program Hypero(17) , and an apparent K(m) for C-CoA was determined to be 12.1 ± 4.8 nM for wild-type and 257 ± 67 nM for S219N. These results obtained for S219N upon addition of long chain acyl-CoA esters supports the conclusion that the non-inducible phenotype was due to reduced affinity for these ligands.


Figure 4: Quenching of intrinsic fluorescence of FadR by acyl-CoA. A, typical fluorescence scan of FadR titrated with C-CoA. FadR alone is labeled 0, and the numbers indicate the nanomolar concentration of C-CoA added in addition to FadR. The data obtained as in A at 339 nm were analyzed using the computer program Hypero(17) . A double-reciprocal plot of data obtained for wild-type FadR (circles) and for S219N (triangles) is given in B. The results are the averages obtained from three experiments. The lines indicate expected values by analysis with Hypero.



Comparison to Other Proteins

Assuming Ser is part of the acyl-CoA binding pocket within FadR, then there are expected to be amino acid identities and structural similarities with other proteins which bind the same or related ligands. Since FadR induction and ligand binding are specific for long chain acyl-CoA esters (C14 or greater), the binding pocket is expected to distinguish both acyl chain length and the coenzyme A moiety, most likely through hydrophobic contacts with the acyl tail and charged residues within the CoA-binding domain. Upon comparison of this region of FadR with proteins which bind acyl-CoA, fatty acids or coenzyme A (using TFASTA and BLAST as well as through literature searching), there was only one protein for which we could detect significant similarities, the CO dehydrogenase/acetyl-coenzyme A synthase (CODH) of C. thermoaceticum.(6) . It was previously determined that tryptophan residue 418 of CODH was specifically protected from modification with 2,4-dinitrophenol sulfenylchloride by CoA(6, 7) . A comparison was made between amino acid residues adjacent to and including Ser of FadR and the region of CODH protected from modification by CoA (Fig. 5). We observed that there are 6 amino acid identities and four conservative substitutions (allowing a 1 amino acid gap in FadR) between the regions encompassing Ser through Arg in FadR and Tyr through Ile in CODH. Trp of CODH, which was identified by 2,4-dinitrophenol sulfenylchloride modification, was among the amino acid identities. These similarities between FadR and CODH suggested that this region of FadR may form all or a portion of the binding site for the CoA moiety of the inducing ligand.


Figure 5: Comparison of the putative CoA binding region of CODH to FadR. The carboxyl-terminal amino acid sequence of FadR from amino acid 213 (14) is compared to amino acids 407-434 of C. thermoaceticum CODH(6) . Amino acids with similar chemical properties are connected by a single line, and identical amino acids are connected with a double line. An asterisk marks Ser in FadR. Trp in CODH which is protected by CoA from chemical modification is maked by a triangle while Trp which is not protected from modification by CoA is marked by a period.



Site-directed Mutagenesis of Amino Acid Residues Adjacent to and Including Ser

To test the contribution of amino acid residues found within the region of FadR which appeared homologous to CODH to induction of FadR by acyl-CoA, we performed site-directed mutagenesis of amino acids 215 through 230 to substitute alanine for the native amino acid. The FadR alleles carrying amino acid substitutions were classified by their relative ability to confer inducible levels of beta-oxidation to the fadR strain LS1346. Individual transformants were grown in TB media or TB media containing 5 mM C (TBO) and in vivo beta-oxidation of 1-[^14C]C was measured. For each experiment, cells transformed with pACYC177 (vector), pCD126 (FadR), and pRW22 (S219N) were included as controls. The results are presented in Table 2. Cells carrying FadR with substitutions of alanine for His, Glu, Ile, His, Arg, and Met had fully inducible levels of beta-oxidation and were classified as wild-type. Alteration of Leu resulted in slightly elevated basal levels of activity as compared with fadR, however transformants of LS1346 carrying the plasmid encoding L230A were not able to grow on minimal media containing decanoate as a carbon source so this allele was also classified as wild-type. Substitutions of alanine for amino acid residues Tyr, Gly, Gln, and Asn appeared to eliminate FadR function as indicated by the constitutive levels of beta-oxidation. Substitutions of alanine for Gly and Trp yielded a strong non-inducible, super-repressor phenotype which was comparable to S219N. LS1346 transformed with plasmid encoding G216A or W223A were not able to grow on media containing long chain fatty acids. A final group of alanine substitutions had induction levels intermediate to wild-type and S219N. These included Glu, Ser, and Lys. Growth on minimal medium containing long chain fatty acids was slowed for cells transformed with plasmids encoding FadR carrying alanine substitutions for these 3 amino acids as compared to wild-type FadR (4 days as compared to 2 days for wild-type FadR). Therefore, these three alleles were designated Weak S to reflect growth and biochemical phenotypes which were intermediate to wild-type and S219N.




DISCUSSION

In these experiments, we used a genetic approach to identify mutations in FadR which resulted in a non-inducible, super-repressor phenotype. Three other FadR alleles which conferred a super-repressor phenotype were previously identified by Hughes et al.(18) . However, the amino acid alterations in those super-repressors encoded within the bacterial chromosome were not defined. The experiments detailed here have identified a region in the carboxyl terminus of FadR which is likely to constitute in part the acyl-CoA binding pocket for this transcriptional regulator. It is expected that binding of long chain acyl-CoA to FadR results in a protein conformation unfavorable for DNA binding causing a cascade of enzyme induction due to the release of FadR-dependent repression of many genes and operons required for growth on fatty acids. Since FadR binding to the fabA promoter activates fabA transcription, binding of acyl-CoA to FadR prevents transcription activation of at least this gene which is required for unsaturated fatty acid biosynthesis. In previous work (2) , we demonstrated that long chain acyl-CoA compounds specifically inhibits DNA binding of FadR. This provided indirect evidence that FadR was able to bind long chain acyl-CoA and that binding of these compounds prevents DNA binding. In the present work, it was shown by isoeletric focusing and by fluorescence quenching that FadR binds long chain acyl-CoA directly.

FadR is a 239 amino acid polypeptide which contains 4 tryptophan residues at positions 21, 60, 75, and 223(14) . Three Trp residues are located in the amino terminus within or very close to the 71 residues believed to comprise the DNA-binding domain (14) and one, Trp, is located within the region identified here as required for inducer binding. The location of the Trp residues within the acyl-CoA binding domain was expected to result in a decrease in steady state intrinsic fluorescence of the protein upon binding ligand. Indeed, this was supported by the substantial degree of quenching of intrinsic fluorescence shown in Fig. 4for FadR upon addition of C-CoA. The magnitude of the decrease in intrinsic fluorescence for FadR upon acyl-CoA binding was somewhat surprising. For yeast N-myristoyl-CoA acyl-transferase, a protein containing 7 Trp residues, only 20% quenching was observed at saturating concentrations of C-CoA(16) . This degree of quenching for N-myristoyl-CoA-acyl-transferase was attributed to a single tryptophan residue. For FadR, the substantial decrease in intrinsic fluorescence upon acyl-CoA binding is likely to be the combined result of acyl-CoA binding to the region including Trp and changes in the environment around Trp residues 21 and 60 and possibly also Trp resulting from an allosteric change which disrupts the DNA binding conformation of the protein. Therefore, the change in fluorescence quenching observed for FadR was not due to the contribution of a single Trp residue as estimated for N-myristoyl-CoA-acyl-transferase but may have been due to the contribution of 2-4 Trp residues in FadR.

The substitution of Ser in FadR with Asn resulted in a non-inducible or super-repressor phenotype. The mutant protein retained the ability to bind DNA and to repress or activate transcription as evidenced by evaluating DNA binding directly and by measuring beta-galactosidase activities in strains carrying fadB-lacZ or fabA-lacZ reporter fusions. The non-inducible phenotype could be due either to a reduced ability to bind long chain acyl-CoA directly or due to an inability to undergo an allosteric transition which disrupts the DNA binding conformation of the protein. These two alternatives are very difficult to distinguish without structural information for the protein bound to either ligand. For example, in LacI substitution of Lys results in a non-inducible phenotype for this repressor(19) . Recently, structural analysis of LacI showed that Lys is surface exposed and does not make direct contact with the ligand. The non-inducible phenotype in the case of LacI is most likely due to conformational perturbations within the quarternary structure of the protein(20) . Two lines of evidence lead us to suggest that the carboxyl-terminal region of FadR is required specifically for binding long chain acyl-CoA, and residues Gly, Ser, and Trp most probably interact with the CoA moiety in particular: (i) The K(m) of binding as estimated by fluorescence quenching or shift in pI differed between the wild-type and the mutant S219N. (ii) This region in FadR, which includes amino acid residues 219-239 of the 239-amino-acid protein shares identities and similarities with CODH of C. thermoaceticum, in a region of CODH for which there is direct biochemical evidence for a CoA-binding site(6, 7) .

Analysis of the set of FadR alleles carrying alanine substitutions also indicates that this region of FadR is critical for function. Alanine substitutions are in general not expected to have severe effects on protein structure since alanine is found throughout protein secondary structures. This has been generally substantiated by mutational analysis(21, 22) . Alanine mutagenesis therefore primarily tests for functional significance of amino acid side chains. Seven substitutions of the 16 tested resulted in a wild-type phenotype. Four substitutions, Y215A, G220A, Q227A, and N229A, resulted in no detectable FadR function indicating that these residues are critical and are perhaps required in particular to maintain the active conformation of the protein. Substitution of Gly or Trp with alanine in FadR resulted in a non-inducible phenotype which was comparable to that of S219N while substitution of Glu, Ser, or Lys with alanine resulted in a reduced level of induction. The clustering of non-inducible, super-repressor mutations in FadR in the COOH terminus provides supportive evidence for function of the region of FadR in binding the inducer, a long chain acyl-CoA. We are subsequently determining the structure of FadR complexed with acyl-CoA to verify and extend this mutational analysis. This protein is of interest as a transcriptional regulator whose activity is modulated by acyl-CoA compounds and due to its unique and complex role as a regulator of many genes encoding proteins required for fatty acid and lipid metabolism.


FOOTNOTES

*
This work was supported by Grant GM38104 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 901-448-8120; Fax: 901-448-7360.

(^1)
The abbreviation used is: CODH, CO dehydrogenase/acetylcoenzyme A synthase.


ACKNOWLEDGEMENTS

We thank Raquel Wilson for initial characterization of pRW22 and Tamra Lynne Heimert for performing the gel shift and beta-galactosidase assays. We are grateful to Dr. Earl Shrago (Department of Nutritional Sciences, University of Wisconsin, Madison) for valuable insights into the similarities between CODH and FadR.


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