(Received for publication, November 13, 1995)
From the
Long chain acyl-acyl carrier protein (acyl-ACP) has been
implicated as a physiological inhibitor of fatty acid biosynthesis
since acyl-ACP degradation by thioesterase overexpression leads to
constitutive, unregulated fatty acid production. The biochemical
targets for acyl-ACP inhibition were unknown, and this work identified
two biosynthetic enzymes that were sensitive to acyl-ACP feedback
inhibition. Palmitoyl-ACP inhibited the incorporation of
[C]malonyl-CoA into long chain fatty acids in
cell-free extracts of Escherichia coli. A short chain acyl-ACP
species with the electrophoretic properties of
-hydroxybutyryl-ACP
accumulated concomitant with the overall decrease in the amount of
[
C]malonyl-CoA incorporation, indicating that
the first elongation cycle was targeted by acyl-ACP. All of the
proteins required to catalyze the first round of fatty acid synthesis
from acetyl-CoA plus malonyl-CoA in vitro were isolated, and
the first fatty acid elongation cycle was reconstituted with these
purified components. Analysis of the individual enzymes and the pattern
of intermediate accumulation in the reconstituted system identified
initiation of fatty acid synthesis by
-ketoacyl-ACP synthase III (fabH) and enoyl-ACP reductase (fabI) in the
elongation cycle as two steps attenuated by long chain acyl-ACP.
Although the steps in the fatty acid biosynthetic pathway are
well characterized, the mechanisms that regulate the production of
fatty acids by the type II, dissociated fatty acid synthase systems
typified by Escherichia coli are largely unknown (for review,
see (1) ). Recent experiments implicate long chain acyl-ACPs ()as feedback inhibitors of the type II pathway. Blocking
phospholipid synthesis at the first acyltransferase step by shifting
either glycerol-phosphate acyltransferase (plsB) or
glycerol-phosphate synthase (gpsA) mutants to the
nonpermissive condition results in the concomitant inhibition of fatty
acid synthesis (2) and the accumulation of long chain
acyl-ACPs(2, 3) . The idea that long chain acyl-ACPs
are feedback regulators of fatty acid biosynthesis arose from
experiments with strains engineered to overexpress thioesterases
capable of degrading acyl-ACP(2, 4) . Strains
overexpressing either of the E. coli thioesterases (tesA or tesB) (2) or a truncated form of tesA that is not exported to the periplasm (4) fail to
accumulate acyl-ACPs when phospholipid production is blocked at the
glycerol-phosphate acyltransferase step and exhibit constitutive fatty
acid synthesis in the absence of phospholipid synthesis. Bacteria
ordinarily cease fatty acid and phospholipid synthesis in stationary
phase, but when acyl-ACP-specific thioesterases cloned from plants are
expressed in E. coli, such strains continue to synthesize and
secrete copious amounts of fatty acids into the medium after growth
ceases(5, 6, 7) . The inhibition of
phospholipid synthesis at the acyltransferase step following the
induction of ppGpp synthesis leads to the accumulation of acyl-ACP and
the concomitant cessation of fatty acid synthesis. Overexpression of
the ppGpp target, plsB, restores phospholipid synthesis,
eliminates the accumulation of acyl-ACP, and relieves the inhibition of
fatty acid synthesis, suggesting that the ppGpp-dependent inhibition of
fatty acid synthesis is a physiological response mediated by
acyl-ACP(8) . Taken together, these in vivo experiments provide compelling evidence that long chain acyl-ACPs
are involved in a regulatory loop that controls the rate of fatty acid
synthesis.
Critically important to the development of this hypothesis is the identification of the enzymatic steps in fatty acid synthesis that are sensitive to acyl-ACP inhibition. The goal of this work was to identify candidate enzymes that are regulated by acyl-ACP.
The fatty acid synthase assay contained 100 µM ACP, 1
mM -mercaptoethanol, 25 µM malonyl-CoA
(specific activity, 57 µCi/µmol), 100 µM acetyl-CoA, 0.1 M sodium phosphate buffer, pH 7.0, 0.1 M LiCl, 1 mM NADH, and 1 mM NADPH in a final
volume of 40 µl. A mixture of ACP,
-mercaptoethanol, and
buffer was preincubated at 37 °C for 30 min prior to the assay to
ensure complete reduction of the ACP, and then the remaining components
(except protein) were added. The mixture was then aliquoted into the
assay tubes and the reaction initiated by the addition of 45 µg of
cell extract protein. The reaction mixture was incubated at 37 °C
for 10 min and the reactions destined for analysis by gel
electrophoresis were stopped in an ice bath. The distribution of
intermediates was determined by conformationally sensitive gel
electrophoresis in 13% polyacrylamide gels containing 0.5 M urea(12, 15) . The amount of fatty acid
synthesized was determined in parallel incubations by adding 20 µl
of 0.1 N NaOH, boiling the sample for 5 min, adding 40 µl
of 0.1 N HCl, and extracting the saponified fatty acids into
hexane. The entire hexane extract was transferred to a scintillation
vial, the hexane evaporated, and the amount of
C-labeled
fatty acids quantitated by scintillation counting.
Figure 1:
Inhibition of long chain
fatty acid formation in vitro by acyl-ACP. A, the
incorporation of [C]malonyl-CoA into long chain
fatty acids was measured using cell extracts as the enzyme and the
saponification/extraction procedure to measure incorporation as
described under ``Experimental Procedures.'' B,
conformationally sensitive gel electrophoresis of the acyl-ACP products
of the reactions shown in A. The acyl-ACP species were
separated using 13% polyacrylamide gels cast in 0.5 M urea,
and the bands were visualized by autoradiography as described under
``Experimental Procedures.''
Figure 2: Inhibition of the individual steps in fatty acid elongation by acyl-ACP. The individual purified enzymes were incubated in the presence or absence of 50 µM palmitoyl-ACP, and the radiolabeled products were separated by conformationally sensitive gel electrophoresis as described under ``Experimental Procedures.''
The inhibition
of the first elongation cycle was examined further by measuring the
effect of increasing concentrations of palmitoyl-ACP on the formation
of butyryl-ACP in the reconstituted system (Fig. 3). These
experiments showed a consistent decrease in butyryl-ACP formation
coupled with increased accumulation of -hydroxybutyryl-ACP as a
function of increasing palmitoyl-ACP in the assay. These data supported
the conclusion that enoyl-ACP reductase (FabI) was an acyl-ACP target.
Furthermore, higher concentrations of palmitoyl-ACP also correlated
with increased accumulation of malonyl-ACP in the assay, consistent
with the conclusion that initiation at the
-ketoacyl-ACP synthase
III (FabH) step is a second point for acyl-ACP regulation of the
pathway.
Figure 3: Dose response for the inhibition of fatty acid synthesis by palmitoyl-ACP. The purified enzymes were incubated in the presence of increasing concentrations of palmitoyl-ACP, and the radiolabeled products were separated by conformationally sensitive gel electrophoresis as described under ``Experimental Procedures.''
Figure 4:
Two sites for the regulation of fatty acid
biosynthesis. The first cycle of fatty acid synthesis is initiated by
-ketoacyl-ACP synthase III (FabH), which condenses acetyl-CoA with
malonyl-ACP to form acetoacetyl-ACP. This intermediate is reduced by
-ketoacyl-ACP reductase (FabG), and the
-hydroxybutyryl-ACP
is dehydrated to crotonyl-ACP by
-hydroxyacyl-ACP dehydrase
(FabA). Butyryl-ACP is formed by the reduction of crotonyl-ACP by
enoyl-ACP reductase (FabI). The direction and thickness of the arrows denote the equilibrium position of each of the
individual reactions and illustrate that FabI pulls the cycle to
completion(15) . Butyryl-ACP is converted to long chain
acyl-ACP through several additional elongation cycles. Acyl-ACPs
regulate fatty acid elongation by feedback inhibition of the elongation
step at FabI and at the initial condensation of acetyl-CoA and
malonyl-ACP by FabH.
-Ketoacyl-ACP synthase III (FabH)
is a second site for the regulation of fatty acid synthesis by acyl-ACP (Fig. 4). Although there is some ambiguity about the pathways
responsible for the initiation of fatty acid synthesis(1) , the
data support the idea that FabH is the primary route in normally
growing cells(13, 14) . Thus, FabH is ideally
positioned in the biosynthetic pathway to regulate initiation and hence
the total number of fatty acids synthesized. A tenable hypothesis for
the biochemical mechanism for acyl-ACP inhibition of FabH is not
readily apparent. FabH differs from the other two condensing enzymes in
that it uses acetyl-CoA rather than acyl-ACP as the primer, so it seems
unlikely that acyl-ACP would inhibit this enzyme by binding to the
acetyl-CoA site. Although the data are consistent with the binding of
acyl-ACP to the malonyl-ACP site, the fact that ACP is not an inhibitor
and the differences in the chemical structures of malonate and long
chain fatty acids open the possibility of a unique acyl-ACP regulatory
site on FabH. Although our experiments illustrate that FabH activity is
inhibited in a coupled enzymatic reaction containing FabD, ACP, and
malonyl-CoA to generate the malonyl-ACP substrate, these mechanistic
questions will only be answered by kinetic analysis of FabH inhibition
by acyl-ACP using purified FabH and malonyl-ACP.
While our present
observations illustrate that the initiation and elongation steps in
fatty acid biosynthesis are subject to regulation by acyl-ACP, two
potential regulatory points related to the production and degradation
of malonyl-CoA could not be tested in our reconstituted system.
Malonyl-CoA does not accumulate when acyl-ACP levels rise in response
to the cessation of phospholipid synthesis in plsB mutants(16) . This observation suggests at first glance
that acetyl-CoA carboxylase is negatively regulated by acyl-ACP.
However, treatment of the glycerol phosphate-starved cells with
cerulenin leads to the accumulation of malonyl-CoA, suggesting that
acetyl-CoA carboxylase is, in fact, operational in the presence of
acyl-ACP. A futile cycle consisting of the transfer of malonyl moieties
from CoA to ACP followed by acyl-ACP-dependent malonyl-ACP
decarboxylation catalyzed by -ketoacyl-ACP synthases I and II and
transacylation of the acetyl-ACP to acetyl-CoA by FabH governs the fate
of malonyl-CoA and may contribute to the down-regulation of fatty acid
synthesis by recycling malonyl-ACP to the acetyl-CoA pool, thus
down-regulating fatty acid elongation and initiation(16) .
Determining the biochemical mechanisms for the regulation of these key
enzymes by acyl-ACP and understanding the relative contributions of
each control point to the physiological regulation of the pathway are
important goals that will be the focus of future research.