(Received for publication, August 10, 1994; and in revised form, October 21, 1994)
From the
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.
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 -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
-C
). 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) ()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.
For
the assay of -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
10
cells/ml, and lysed by sonication.
-Galactosidase activity was assayed immediately using o-nitrophenyl-
-D-galactopyranoside as substrate
as described by Miller(10) . Specific activities were
calculated using a value of 1.25 µg of protein/Klett unit.
To assess inducibility, plasmid DNA was
transformed into strain LS1346 (fadR) and in vivo rates of -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
-oxidation, which were induced 2-3-fold
as compared with 18-fold for wild-type FadR. These low levels of in
vivo
-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.
-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.
Figure 1:
DNA binding affinity of wild-type
and S219N FadR proteins. Gel shift experiments were performed using
purified FadR and 1 10
DNA containing
O
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
10
[
P]O
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
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
was
determined for C
-CoA.
Figure 2:
Inhibition of DNA binding by
C-CoA. Gel shift analysis was performed with DNA
containing O
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.
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
-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
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.
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.
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
-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
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.