The biochemical mechanisms responsible for the regulation of the
type II, dissociated fatty acid synthase systems typified by Escherichia coli is an area under active investigation (for
reviews, see (1) and (2) ). Several lines of evidence
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-P acyltransferase (plsB) or glycerol-P synthase (gpsA) mutants to the
nonpermissive condition results in the concomitant inhibition of fatty
acid synthesis (3) and the accumulation of long-chain
acyl-ACPs(3, 4) . Furthermore, 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 mediated by acyl-ACP(5) . The idea that
long-chain acyl-ACPs are feedback regulators of fatty acid biosynthesis
was directly tested in strains engineered to overexpress thioesterases
capable of degrading acyl-ACP(3, 6) . Strains
overexpressing either of the E. coli thioesterases (tesA or tesB) (3) or a truncated form of tesA that is not exported to the periplasm (6) fail to
accumulate acyl-ACP when phospholipid production is blocked at the
glycerol-phosphate acyltransferase step, resulting in constitutive
fatty acid synthesis in the absence of phospholipid synthesis.
Normally, bacteria in stationary phase down-regulate fatty acid and
phospholipid synthesis, but when acyl-ACP-specific thioesterases
derived from plants are expressed in E. coli, such strains
continue to synthesize and secrete copious amounts of fatty acid into
the medium after growth ceases(7, 8, 9) .
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 and hence phospholipid
formation.
-Ketoacyl-ACP synthase III is implicated as a
regulatory site based on the accumulation of malonyl-ACP following the
inhibition of a reconstituted fatty acid synthase system by long-chain
acyl-ACP(10) .
-Ketoacyl-ACP synthase III catalyzes the
condensation of acetyl-CoA with malonyl-ACP to initiate new rounds of
chain elongation and is therefore ideally positioned in the pathway to
regulate the overall rate of fatty acid
synthesis(11, 12) . Synthase III is distinct from the
other two condensing enzymes in that it is not inhibited by the
antibiotic cerulenin and specifically uses acetyl-CoA, rather than
acyl-ACP as a primer(11, 12) . Synthase III (FabH) (
)is the product of the fabH gene, which is
localized within a cluster of fatty acid biosynthetic genes at minute
24.5 on the E. coli chromosome (13) . The DNA sequence
predicts a protein with a molecular mass of 33.5 kDa, containing an
active site cysteine embedded within a sequence characteristic of other
condensing enzyme active sites(13) . A second region of the
protein has a high degree of similarity to a motif found in proteins
that bind CoA thioesters. Overproduction of FabH protein leads to a
decrease in the overall chain-length of fatty acids in the membrane
phospholipids consistent with its proposed role in the initiation of
fatty acid biosynthesis(13) . However, basically nothing is
known about the substrate specificity or regulatory properties of FabH.
The focus of this study is to biochemically characterize FabH and
define the kinetic mechanism that underlies the negative regulation of
synthase III activity by long-chain acyl-ACP.
EXPERIMENTAL PROCEDURES
Materials
Sources of supplies were: Moravek
Biochemicals Inc., [2-
C]malonyl-CoA (specific
activity, 57 mCi/mmol) and [1-
C]acetyl-CoA
(specific activity, 54 mCi/mmol); Sigma, fatty acids, ACP, cerulenin,
and acyl-CoAs; Promega, molecular biology reagents; Novagen, pET
vectors and expression strains; Qiagen, Ni
-agarose
column; and Pharmacia Biotech Inc., acetyl-CoA and malonyl-CoA.
Acyl-ACPs were synthesized using the acyl-ACP synthetase purified from
an overproducing strain (14) as described
previously(15) . The acyl-ACPs were concentrated, and the
buffer exchanged, by centrifugal filtration in a Centricon-3
concentrator (Amicon). Yields were judged by Bradford protein
determination (16) and conformationally sensitive gel
electrophoresis in a 2.5 M urea, 13% acrylamide
gel(17) . All other chemicals were of reagent grade or better.
Coupled Assay of FabH
The synthase III (FabH)
assay contained 22 µM ACP, 1 mM
-mercaptoethanol, 65 µM malonyl-CoA, 45
µM [1-
C]acetyl-CoA (specific
activity 56 µCi/µmol), FabD (0.2 µg of protein), 0.1 M sodium phosphate buffer, pH 7.0, and 0.02-0.2 µg of
protein 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 FabH) were added. The mixture was
then aliquoted into the assay tubes and the reaction initiated by the
addition of FabH. The reaction mixture was incubated at 37 °C for
12 min, and then 35 µl was removed and deposited on a Whatman No.
3MM filter disc. The disc was washed with three changes (20 min each)
of ice-cold trichloroacetic acid. The concentration of the
trichloroacetic acid was reduced from 10 to 5 to 1% in each successive
wash. The filters were dried and counted in 3 ml of scintillation
fluor.
Malonyl-CoA:ACP Transacylase Assay
The malonyl
transacylase (FabD) assay contained 100 µM ACP, 1 mM
-mercaptoethanol, 0.1 M sodium phosphate, pH 7.0, 45
µM [2-
C]malonyl-CoA (specific
activity 57 mCi/mmol). The ACP was reduced by incubation in the buffer
plus
-mercaptoethanol for 30 min at 37 °C prior to addition to
the assay tube, and the reaction was initiated by the addition of
[2-
C]malonyl-CoA. The amount of
[2-
C]malonyl-ACP formed was quantitated using
the same filter disc assay described above for FabH.
Construction of Expression Vectors and Purification of
the His-tagged Proteins
The genes were amplified from genomic
DNA obtained from strain UB1005. Primers created novel restriction
sites for NdeI at the N-terminal methionine and BamHI
downstream of the stop codon. Polymerase chain reaction was performed
with Taq DNA polymerase, and the fragments ligated into the TA
cloning vector pCRII (Invitrogen) and transformed into E. coli OneShot cells. Following overnight growth, plasmid was isolated
and digested with NdeI and BamHI, and the appropriate
fragments isolated and ligated into NdeI- and BamHI-digested pET-15b. These mixtures were used to transform
strain BL21(DE3) to ampicillin resistance, and resultant colonies were
screened for their ability to overexpress proteins of the correct size
following isopropyl-1-thio-
-D-galactopyranoside
induction. One such clone was chosen for each gene and grown in 100 ml
of M9 medium (18) supplemented with 1% casein amino acids,
0.4% glucose, and 1 mM MgCl
to a density of
approximately 5
10
cells/ml.
Isopropyl-1-thio-
-D-galactopyranoside was then added to a
final concentration of 1 mM, and incubation continued for an
additional 3 h at 37 °C. Cells were collected by centrifugation
(8,000 rpm, 4 °C, 10 min), and stored at -20 °C
overnight. Lysis was effected according to (19) , with the
addition of lysozyme to 0.1 mg/ml after resuspension. Soluble protein
was applied to a Ni
-agarose column and washed
with 40 mM imidazole-containing metal-chelation affinity
chromatography buffer (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 1 mM
-mercaptoethanol). His-tagged proteins
were eluted with 200 mM imidazole in the same buffer and the
purified proteins stored at -20 °C.
Preparation of Malonyl-ACP
Malonyl-ACP was
prepared using purified histidine-tagged malonyl-CoA:ACP transacylase
(His-tag-FabD) in a reaction containing 100 µM ACP, 1
mM
-mercaptoethanol, 0.1 M sodium phosphate, pH
7.0, 200 µM malonyl-CoA, and 250 µg of enzyme in a
total volume of 10 ml. The reaction was incubated at 37 °C for 30
min, then applied to a 3-ml anion-exchange (DE-52) column
pre-equilibrated with 20 mM Tris-HCl, pH 7.4, 1 mM
-mercaptoethanol. Reaction components were removed by washing
the column with 15 ml of 0.25 M LiCl in the equilibration
buffer. Malonyl-ACP was then eluted with 0.45 M LiCl in the
equilibration buffer. The protein-containing fractions were pooled and
exchanged into the equilibration buffer without LiCl with a Centricon-3
microconcentrator. Conversion of ACP to malonyl-ACP was determined by
conformationally sensitive gel electrophoresis in a 0.5 M urea, 13% acrylamide gel.
Biochemical Characterization of Purified
FabH
Synthase III activity of the purified His-tagged FabH was
assayed in a reaction mixture containing 30 µM malonyl-ACP, 200 µM [
C]acetyl-CoA (specific activity, 27
mCi/mmol), 0.1 M sodium phosphate, pH 7.0, 1 mM
-mercaptoethanol, and acyl-ACP as indicated. Reactions were
initiated by the addition of 0.04 µg of purified protein, and
incubated at 37 °C for 12 min. Incorporation of label was stopped
by transferring 35 µl of the reaction onto a Whatman No. 3MM filter
paper disc, followed by washing in trichloroacetic acid and
scintillation counting as described above. Kinetics were established by
varying the concentration of malonyl-ACP or acetyl-CoA at different
fixed concentrations of acyl-ACP. The transacylase activity of FabH was
measured in assays containing 200 µM [
C]acetyl-CoA (specific activity, 27
mCi/mmol), 0.1 M sodium phosphate, pH 7.0, 100 µM ACP, 1 mM
-mercaptoethanol, and 0.2-2.4 µg
of enzyme. Reactions were incubated at 37 °C for 12 min, and the
amount of [
C]acetyl-ACP formed was determined
using the filter disc assay described above. Reactions were linear with
time and protein.The chain-length specificity of FabH for acyl-CoA
primers was determined in a coupled system containing FabH, FabD, and
-ketoacyl-ACP reductase (FabG) using the reaction conditions
described previously(20) . The purified enzymes (0.07
µg/reaction) were incubated in 0.1 M sodium phosphate, pH
7.0, containing 10-160 µM of acyl-CoA (chain lengths
2-8), 1 mM
-mercaptoethanol, 50 µM ACP, 100 µM NADPH, and the reactions initiated by the
addition of 25 µM [2-
C]malonyl-CoA
(specific activity 57 mCi/mmol) in a final volume of 40 µl. The
reactions were incubated for 12 min at 37 °C and stopped by placing
in an ice slurry. The products of the reactions were analyzed by
separation on 13% polyacrylamide gels containing 0.5 M urea,
followed by autoradiography.
RESULTS AND DISCUSSION
Acyl-ACPs Inhibit
-Ketoacyl-ACP Synthase III
(FabH) in Vitro
Our previous work indicating FabH as a target
for acyl-ACP regulation was based on a decrease in acetoacetyl-ACP
formation and the accumulation of malonyl-ACP using an in vitro assay that relied on conformationally sensitive gel
electrophoresis to analyze the products(10) . The drawback to
the conformationally sensitive gel electrophoresis assay is that the
recovery of less stable ACP thioesters, such as malonyl-ACP and
acetoacetyl-ACP, is reduced by the high pH and urea concentrations
required to achieve the separations. Thus, we tested the ability of
long-chain acyl-ACP to inhibit the activity of synthase III in a
coupled assay system. The malonyl-ACP substrate was generated by the
inclusion of malonyl-CoA, ACP, and malonyl-CoA:ACP transacylase (FabD)
in the assay. [1-
C]Acetyl-CoA was the other
substrate. Long-chain acyl-ACPs (16:0- or 18:1
11-ACP) were potent
inhibitors of FabH activity (data not shown). To verify that the
inhibition was not due to the inactivation of malonyl transacylase, a
specific assay for FabD activity was performed using
[2-
C]malonyl-CoA as the substrate. Long-chain
acyl-ACPs did not inhibit the formation of malonyl-ACP (data not
shown). In fact, the addition of acyl-ACPs resulted in a modest
stimulation of FabD activity. The acyl-ACP preparations used in these
experiments were approximately >95% pure, and the slight elevation
of FabD activity by acyl-ACP was attributed to the presence of small
amounts of contaminating ACP in the acyl-ACP preparations since the
stimulatory effect was mimicked by adding ACP to the assay at 5% of the
concentration of acyl-ACP (not shown). These data corroborated our
previous conclusion that FabH was a target for acyl-ACP inhibition and
encouraged us to synthesize malonyl-ACP and a spectrum of acyl-ACPs to
determine the biochemical mechanism for FabH regulation using defined
assay components.
Purification of FabH and Preparation of
Substrates
To directly investigate the regulation of FabH
activity by acyl-ACP, a collection of purified proteins and ACP
thioesters was required (Fig. 1). First, a His-tag-FabH fusion
protein expression vector was constructed and the FabH enzyme was
purified to homogeneity by metal chelate column chromatography (Fig. 1C). Second, a His-tag-FabD fusion protein
expression vector for malonyl transacylase (FabD) was constructed and
this enzyme was also purified to homogeneity by metal chelate column
chromatography (Fig. 1C). The FabD enzyme was used to
synthesize malonyl-ACP from malonyl-CoA. Conditions were established
that gave nearly complete conversion of ACP to malonyl-ACP, which was
isolated by ion-exchange chromatography and desalted for use in the
FabH assay (Fig. 1A). The long-chain acyl-ACPs were
>95% pure as judged by conformationally sensitive gel
electrophoresis, with the exception of 20:0-ACP, which was only 50%
pure (Fig. 1B).
Figure 1:
Preparation and
purity of enzymes, substrates, and inhibitors. Panel A,
conformationally sensitive gel electrophoresis of malonyl-ACP
synthesized using the purified His-tag-FabD protein. Panel B,
conformationally sensitive gel electrophoresis of the acyl-ACPs used in
this study synthesized using the acyl-ACP synthetase method. Panel
C, SDS gel electrophoresis of purified His-tag-FabH and
His-tag-FabD. Details of the purification schemes and the different
electrophoretic conditions used to assess the purity of the reagents
are described under ``Experimental
Procedures.''
Kinetic Constants for Acetyl-CoA and
Malonyl-ACP
The acetoacetyl-ACP synthase specific activity of
the purified His-tag-FabH protein was 3.57 nmol/min/µg.
Acetyl-CoA:ACP transacylase activity was also present in this
preparation at a specific activity of 13 pmol/min/µg. This ratio of
activity is the same as we reported previously for purified,
recombinant FabH when assayed using a coupled enzyme system (13) and indicated that the histidine tag did not alter the
biochemical properties of the enzyme. The apparent K
values for acetyl-CoA and malonyl-ACP were determined to be 40
and 5 µM, respectively (Fig. 2). The size of the
intracellular CoA thioester pool varies with the carbon source used for
growth, and higher concentrations generally correlate with increased
growth rate(21) . The acetyl-CoA concentration is 325
µM in cells growing in glucose minimal medium, whereas
malonyl-CoA (
2 µM) comprises a small fraction of the
total CoA thioester pool(21) . The total ACP pool is maintained
at approximately 15% of the CoA pool (21, 22, 23) and malonyl-ACP is estimated to
be between 10 and 30% of the total ACP pool comparing measurements made
in several different papers (11, 12, 24) .
Therefore, the affinity of FabH for its two substrates reflects the
differences in the relative abundance of acetyl-CoA and malonyl-ACP in vivo.
Figure 2:
Kinetic constants for FabH. The apparent K
values for acetyl-CoA (panel
A) and malonyl-ACP (panel B) were determined using
purified His-tag-FabH and malonyl-ACP and the assay conditions
described under ``Experimental
Procedures.''
Primer Specificity
Our previous work with FabH
indicated that this enzyme utilized acetyl-CoA, but not acetyl-ACP, as
the primer for the condensation reaction(12) . This result was
verified using the purified His-tagged FabH protein (data not shown).
We determined the acyl chain specificity for CoA thioesters as primers
in the FabH reaction (Fig. 3). We did not have ready access to
C-labeled acyl-CoAs for each chain length; therefore, the
FabH assay was modified to label the products with
[2-
C]malonyl-CoA and quantitate product
formation by conformationally sensitive gel electrophoresis. Because
-ketoacyl-ACP thioesters are not stable to the electrophoretic
conditions employed, we also included
-ketoacyl-ACP reductase
(FabG) and NADPH to the reactions to convert the unstable
-ketoacyl-ACPs to their corresponding stable
-hydroxyacyl-ACPs for analysis. As anticipated, this system
efficiently converted acetyl-CoA to
-hydroxybutyryl-ACP. FabH was
also able to use propionyl-CoA and butyryl-CoA as primers, although the
efficiency of condensing butyryl-CoA with malonyl-ACP was significantly
lower than acetyl-CoA and propionyl-CoA. FabH was unable to utilize
either hexanoyl-CoA or octanoyl-CoA as primers (data not shown).
Figure 3:
FabH acyl-CoA primer specificity.
Reactions containing FabH, FabD, and FabG were performed using the
indicated amount of acyl-CoA primer (10-160 µM) and
[2-
C]malonyl-ACP as described under
``Experimental Procedures.'' The reaction mixtures were
separated by conformationally sensitive gel electrophoresis and the
formation of the
-hydroxyacyl-ACPs was determined by
autoradiography. Abbreviations are: Mal-ACP, malonyl-ACP; Ac-ACP, acetyl-ACP;
-OH 4:0-ACP,
-hydroxybutyryl-ACP;
-OH 5:0-ACP,
-hydroxypentanoyl-ACP;
-OH 6:0-ACP,
-hydroxyhexanoyl-ACP.
A
minor band that co-migrated with acetyl-ACP was also observed, and the
amount of this product was inversely proportional to the amount of
-hydroxyacyl-ACP formed, and most prevalent in assays that lacked
an acyl-CoA primer (data not shown). Acetyl-ACP formation is most
notable at 10 and 20 µM primer concentration when
butyryl-CoA was used as substrate (Fig. 3). Acetyl-ACP is formed
either by the decarboxylation of malonyl-ACP or acetyl transfer from
acetyl-CoA to ACP(1, 2) . This latter reaction,
however, would not lead to labeled acetyl-ACP, since
[
C]malonyl-ACP was used as the substrate in
these assays. Similar experiments performed with
[
C]acetyl-CoA in the absence of added
malonyl-CoA did not lead to the appearance of the band corresponding to
acetyl-ACP. The only requirements for the formation of acetyl-ACP were
FabD, FabH, and [
C]malonyl-CoA; therefore, we
assigned the production of acetyl-ACP to the FabH-catalyzed
decarboxylation of malonyl-ACP in the absence of a suitable acyl-CoA
primer. However, the intracellular concentrations of acetyl-CoA are
large compared to malonyl-CoA and malonyl-ACP; therefore, it is
unlikely that the decarboxylation of malonyl-ACP by FabH is a
significant reaction during logarithmic growth.
These data are
consistent with the proposed role of FabH in the initiation of fatty
acid synthesis. E. coli efficiently employs exogenous
propionate as a primer to form fatty acids with odd numbers of carbon
atoms(25) , and our results indicate that FabH represents an
efficient pathway for the incorporation of propionate into fatty acids.
Treatment of E. coli with the antibiotic cerulenin, which
inactivates synthases I and II(1) , leads to the accumulation
of butyryl-ACP and other short-chain
acyl-ACPs(11, 20, 24) . One interpretation of
this result is that FabH is capable of using short-chain acyl-ACPs;
however, we have never been able to demonstrate that FabH is capable of
even a small rate of utilization of acetyl-ACP (12) or that it
is able to elongate butyryl-ACP(20) . Another possibility is
that there is another cerulenin-resistant condensing enzyme.
Siggaard-Anderson and co-workers (26) conclude that they have
detected and cloned a fourth condensing enzyme. However, these
conclusions appear to be in error, since it is clear that their clone
actually encodes condensing enzyme II (fabF) (27) and
the distribution of products in their assays can be explained by the
presence of a mixture of incompletely separated condensing enzymes in
the column fractions. Our data suggest an additional interpretation.
Short-chain acyl-ACPs that accumulate in cerulenin-treated cells may
arise from FabH utilizing butyryl-CoA to prime fatty acid biosynthesis.
There are no data available on the cellular concentration of
butyryl-CoA or the potential significance of this thioester as a primer
for fatty acid synthesis in E. coli.
Acyl-ACP Inhibition of FabH Activity
Long-chain
acyl-ACP inhibited FabH enzyme in vitro, whereas similar
concentrations of ACP were without effect (Fig. 4). The potency
of the acyl-ACPs increased with increasing chain length between 12 and
20 carbon atoms, and the unsaturated 18:1
11-ACP was consistently
more effective than the corresponding saturated acyl-ACP (18:0-ACP).
The kinetic mechanism for the acyl-ACP inhibition was examined with
respect to both acetyl-CoA and malonyl-ACP (Fig. 5). The mode of
inhibition with respect to acetyl-CoA was mixed, i.e. a
combination of competitive and noncompetitive inhibition (Fig. 5A). The pattern of inhibition with respect to
malonyl-ACP was competitive. Condensing enzymes proceed by an ordered
reaction mechanism with the formation of the acyl-enzyme intermediate
from the primer preceding the binding of malonyl-ACP and the subsequent
condensation reaction(28, 29) . The mixed type of
inhibition with respect to acetyl-CoA was consistent with the acyl-ACP
inhibitor binding to both the free enzyme and to the acyl-enzyme
intermediate (Fig. 6). Thus, inhibition was competitive with
respect to malonyl-ACP, which only bound to the acyl-enzyme
intermediate. However, inhibition was competitive with respect to
acetyl-CoA when acyl-ACP bound to the free enzyme and noncompetitive
with repect to acetyl-CoA when acyl-ACP bound to the acyl-enzyme
intermediate.
Figure 4:
Chain-length specificity for the
inhibition of FabH activity by acyl-ACP. FabH activity was assayed
using the standard conditions for purified FabH as described under
``Experimental Procedures'' in the presence of 100 µM of the indicated acyl-ACP. Data are the averages from two
experiments.
Figure 5:
Kinetics for acyl-ACP inhibition of FabH
activity. Panel A, mixed inhibition of FabH activity by
16:0-ACP with respect to acetyl-CoA. Panel B, competitive
inhibition of FabH activity by 16:0-ACP with respect to malonyl-ACP.
Details of the assay conditions used for the kinetic analysis are
described under ``Experimental
Procedures.''
Figure 6:
Proposed mechanism for the inhibition of
FabH by acyl-ACP. The FabH catalytic cycle begins by the transfer of
the acetyl moiety from acetyl-CoA to the active site sulfhydryl
(Cys-112) to form an acyl-enzyme intermediate. The acyl-enzyme
intermediate then reacts with malonyl-ACP to release acetoacetyl-ACP
and CO
. Acyl-ACP inhibits the reaction by binding to either
the free enzyme or the acyl-enzyme
intermediate.
Acyl-ACP and Malonyl-ACP Concentrations in
Vivo
The intracellular CoA and ACP concentrations vary depending
on the stage of growth and the carbon
source(21, 22, 23, 30, 31) .
Estimated concentrations of CoA range from 0.11 to 1 mM, and
ACP varies between 17 and 130 µM. The important issue is
not the absolute concentration of acyl-ACP attainable in the cell, but
how the ratio of malonyl-ACP to long-chain acyl-ACP changes during
regulatory responses since acyl-ACP inhibition of FabH is competitive
with respect to malonyl-CoA. We examined these parameters in strain
SJ22 (panD2 plsB26) before and after starvation for glycerol-P
to block phospholipid synthesis, accumulate acyl-ACP, and inhibit fatty
acid synthesis(24) . As observed
previously(4, 24) , the removal of glycerol-P
triggered the accumulation of long-chain acyl-ACP (Fig. 7). The
cells were grown to mid-logarithmic phase on 0.5 µM
-[3-
H]alanine, and the levels of CoA
(900 µM) and ACP (114 µM) were estimated from
the recovery of label in the two fractions, the cell number, and an
estimate of the cell volume of 0.437 µl/5
10
cells (21) . The percentage of the individual components
of the ACP pool was determined by densitometry of the gels shown in Fig. 7. Malonyl-ACP was approximately 19% of the total (21
µM) and decreased to 8% of the pool (9 µM)
following glycerol-P starvation (Fig. 7A). Long-chain
acyl-ACPs were essentially not detected prior to glycerol-P starvation
but rose to 53.5% of the total (61 µM) after removal of
glycerol-P (Fig. 7B). The acyl-ACP:malonyl-ACP ratio
was 6.3 in the starved cells compared to 3.3 in the experiments
depicted in Fig. 4. These calculations indicate that acyl-ACP
regulation is a plausible mechanism for controlling FabH activity in vivo.
Figure 7:
Composition of the ACP pool following
cessation of phospholipid synthesis. Strain SJ22 (panD2 plsB26
plsX50) (4) was grown in M9 minimal medium supplemented
with glucose (0.4%), casein hydrolysate (0.1%), thiamine (0.001%),
glycerol-P (0.04%), and
-[3-
H]alanine (0.5
µM, specific activity 91 Ci/mmol). At a density of 5
10
cells/ml, the culture was split and the
glycerol-P was removed from half. The control and glycerol-P-deprived
cells were incubated for 10 min at 37 °C, the cells were
harvested,and the ACP extracted(20) . The percentage of the
pool corresponding to malonyl-ACP and long-chain acyl-ACP in the
presence and absence of glycerol-P was determined by conformationally
sensitive gel electrophoresis using 13% polyacrylamide gels containing
either 0.5 M urea (panel A) or 2.5 M urea (panel B). The bands were visualized by fluorography and the
relative amount of ACP in each band determined by
densiometry(20) .
Conclusions
The inhibition of
-ketoacyl-ACP
synthase III activity by acyl-ACP reveals a role for FabH in the
feedback regulation of fatty acid synthesis. Since FabH catalyzes the
first condensation step in fatty acid biosynthesis, it is ideally
positioned in the pathway to control the rate of fatty acid initiation
and hence the total amount of fatty acids produced. The increasing
potency of acyl-ACP inhibition of FabH activity with increasing acyl
chain length is consistent with the in vivo observations.
Fatty acid biosynthesis is most stringently regulated when phospholipid
synthesis is curtailed at the glycerol-phosphate acyltransferase
step(3, 24) , supporting the idea that the long-chain
acyl-ACPs are the most potent physiological regulators of fatty acid
synthesis. Jiang and Cronan (3) indicate that starvation of a
glycerol-P auxotroph triggers the accumulation of a predominant
acyl-ACP species possessing the electrophoretic properties expected for
20:1
13-ACP. Although our findings point to a role for FabH in this
regulatory scheme, it seems clear that the control of other pathway
enzymes also contributes to the physiological regulation of fatty acid
production by acyl-ACP. Regulation at the acetyl-CoA carboxylase and/or
acyl-ACP-dependent malonyl-ACP decarboxylation are two steps that are
also likely to contribute to the attenuation of fatty acid synthesis by
acyl-ACP(24) . Enoyl-ACP reductase (fabI) is inhibited
by acyl-ACP (10) and plays a determinant role in completing
rounds of fatty acid elongation (20) indicating that the
biochemical mechanism underlying the regulation of this enzyme by
acyl-ACP should be investigated.