The fatty acid synthase system of Escherichia coli is
the paradigm for the type II or dissociated fatty acid synthase systems
(for review see (1) ). Each of the individual enzymes are
encoded by distinct genes, and the same chemical reaction is often
catalyzed by multiple isozymes. There are four basic reactions that
constitute a single round of elongation (Fig. 1). The first step
is condensation of malonyl-ACP (
)with either acetyl-CoA to
initiate fatty acid synthesis or the growing acyl chain to continue
cycles of elongation. The first cycle of elongation is initiated by the
condensation of acetyl-CoA with malonyl-ACP catalyzed by
-ketoacyl-ACP synthase III (FabH), the product of the fabH gene. Subsequent cycles of elongation are catalyzed by condensing
enzyme I (FabB) or II (FabF). Recently, the existence of a fourth
condensing enzyme was proposed(2) . However, the map position
and sequence of this putative new enzyme are the same as that of the fabF gene(3) ; therefore there is little concrete
evidence for the presence of a fourth condensing enzyme. The
-ketoacyl-ACP is reduced by a NADPH-dependent
-ketoacyl-ACP
reductase (FabG). Only a single enzyme is known to be responsible for
this step, although the existence of other isozymes cannot be ruled
out. There are two
-hydroxyacyl-ACP dehydrases (FabA and FabZ)
capable of forming trans-2-enoyl-ACP. The product of the fabA gene is specifically involved in the introduction of a cis-double bond into the growing acyl chain at the
-hydroxydecanoyl-ACP step. The spectrum of reactions catalyzed by
the FabZ dehydrase is unknown, but the fact that mutations in the fabZ gene were isolated based on their ability to suppress the
temperature-sensitive growth phenotype in lpxA2(Ts)
(
-hydroxymyristoyl-ACP:UDP-N-acetylglucosamine
acyltransferase) mutants indicates that it plays an important role in
long-chain saturated fatty acid formation(4) . The last
reaction in each elongation cycle is catalyzed by enoyl-ACP reductase
(FabI). E. coli is thought to possess two enoyl-ACP
reductases, one NADH-dependent and the other
NADPH-dependent(5) , suggesting that more than one isozyme
catalyzes this step of fatty acid biosynthesis.
Figure 1:
Cycles of fatty
acid elongation are pulled by enoyl-ACP reductase (FabI). There are
four reactions in each cycle of fatty acid elongation. The first step
is catalyzed by
-ketoacyl-ACP synthase, which condenses
malonyl-ACP with either acetyl-CoA (synthase III, FabH) or acyl-ACP
(synthases I and II, FabB, and FabF, respectively). The malonyl-ACP
used in the condensing enzyme step is formed by malonyl-CoA:ACP
transacylase (FabD). Reduction of the ketoester is catalyzed by the
NADPH-dependent
-ketoacyl-ACP reductase (FabG). The third step is
catalyzed by
-hydroxyacyl-ACP dehydrase (either FabA or FabZ). The
final step in each cycle is catalyzed by the NADH-dependent enoyl-ACP
reductase (FabI), which converts trans-2-enoyl-ACP to
acyl-ACP. The inner arrows indicate the overall direction of
the cycle in fatty acid biosynthesis. The outer thick arrows diagrammatically indicate the equilibrium positions for the
enzymatic reactions determined in this
study.
The gene encoding
the NADH-dependent enoyl-ACP reductase (fabI) was discovered
as an outgrowth of research on the mechanism of action of diazaborines,
a class of heterocyclic antibiotics. Biosynthesis of fatty acids and
phospholipids is inhibited when E. coli or Salmonella
typhimurium are treated with diazaborines, and resistance to these
antibiotics is associated with an allelic form of the envM gene product (6) . The envM gene was first
identified as a temperature-sensitive mutation with an
osmotically-repairable membrane defect(7) . Because the EnvM
protein, purified from an overproducing strain, possesses
NADH-dependent enoyl-ACP reductase activity and binds radiolabeled
diazaborine, the gene was renamed fabI(8) . The fabI (envM) gene has been sequenced, and the
diazaborine resistance is associated with a point mutation in this
locus(9) . Similarly, the product of the fabI analog
in Mycobacterium tuberculosis, the inhA gene product,
is the target for the antibiotics isoniazid and ethionamide, and a
single point mutation in the inhA gene confers resistance to
both antibiotics in M. tuberculosis(10) . InhA has
recently been purified, crystallized, and shown to be an NADH-dependent
enoyl-ACP reductase(11) . The focus of this study is to
determine the number of enoyl-ACP reductase enzymes in E. coli and to define the role of enoyl-ACP reductase in regulating cycles
of fatty acid elongation.
EXPERIMENTAL PROCEDURES
Materials
-[3-
H]Alanine
(specific activity, 56 Ci/mmol) and En
Hance were purchased
from DuPont NEN. [2-
C]Malonyl-CoA (specific
activity, 57 mCi/mmol) was purchased from Moravek Biochemicals Inc.
Cerulenin, tetradecanoic acid, and ACP were purchased from Sigma. The cis-7-tetradecenoic acid was the generous gift of Dr. John E.
Cronan, Jr. Fatty acids were enzymatically coupled to ACP (12, 13) and then concentrated, and the salt was
removed by centrifugal filtration in a Centricon-3 concentrator
(Amicon). Protein amounts were assayed by the Bradford
method(14) , and purity was estimated by electrophoresis in a
2.5 M urea, 15% acrylamide gel(15) . Acetyl-CoA and
malonyl-CoA were from Pharmacia Biotech Inc. All other chemicals were
of reagent grade or better.
In Vivo Labeling of the ACP Pool
Strain
RJH13 (panD fabI(Ts)) (16) was grown at 30 °C to a
density of 5
10
cells/ml in M9 medium (17) supplemented with 0.4% glucose and 0.5 µM
-[
H]alanine (specific activity, 56 Ci/mmol),
at which time an aliquot of the culture was removed and placed into a
flask prewarmed to 42 °C. Samples (0.5 ml) were taken after 10 min
of incubation, and cerulenin (1 mg/ml) was added to the remaining
portions and incubated for a further 10 min. Samples were immediately
pelleted by centrifugation in a microfuge (15,000 rpm, 4 °C, 3 min)
and then lysed by a procedure using successive treatments with sucrose,
lysozyme, EDTA, and Triton X-100(18) . Products were analyzed
by conformationally sensitive electrophoresis in 13% polyacrylamide
gels containing 0.5 M urea (15, 16, 19) , and the bands were visualized
by fluorography. Strain SJ16 (panD) (20) was grown at
37 °C to a density of 5
10
cells/ml, and the
culture was split; one half was treated with 1 mg/ml cerulenin for 10
min, and the other half was harvested as a control.
Preparation of Cell-free Extracts
The
crude fatty acid synthase preparation and the fabI(Ts) extract
were obtained as described(21) . Briefly, a 500-ml culture of
either E. coli strain UB1005 or RJH13 (fabI(Ts)) was
grown to late log phase in LB medium, and the cells were harvested by
centrifugation. Cells were resuspended in 5 ml of lysis buffer (0.1 M sodium phosphate, pH 7.0, 5 mM
-mercaptoethanol, 1 mM EDTA) and disrupted by
passage through a French pressure cell at 18,000 psi. The lysate was
centrifuged in a JA-20 rotor at 20,000 rpm at 4 °C for 1 h to
remove cell debris, and protein in the 45-80% ammonium sulfate
cut of the supernatant was collected. This pellet was resuspended in 2
ml of lysis buffer and dialyzed overnight at 4 °C against 1 liter
of lysis buffer. Protein was determined by the Bradford
method(14) .
Construction of Expression Vectors and Purification
of the His-Tag 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 (Promega), and the
fragments were ligated into the TA cloning vector pCRII (Invitrogen)
and transformed into E. coli OneShot cells (Invitrogen).
Following overnight growth, plasmid was isolated and digested with NdeI and BamHI, and the appropriate fragments were
isolated by GeneClean (Bio 101, Inc.) and ligated into NdeI
and BamHI-digested pET-15b (Novagen). These mixtures were used
to transform strain BL21(DE3) (Novagen) 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 (17) supplemented with 1% casamino acids (Difco), 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 was continued for
a further 3 h at 37 °C. Cells were collected by centrifugation in a
JA-20 rotor (8,000 rpm, 4 °C, 10 min), and stored at -20
°C overnight. Lysis was affected according to (22) , with
the addition of lysozyme to 0.1 mg/ml after resuspension. Soluble
protein was applied to a Ni
-agarose column (QIAGEN)
and washed with 40 mM imidazole in a metal-chelation affinity
chromatography buffer (20 mM Tris-Cl, 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 were stored at -20 °C.
Enzyme Assays
To assay for reduction of
short chain enoyl-ACPs, reactions (40 µl) contained 50 µM acetyl CoA, 25 µM ACP, 0.1 M sodium
phosphate, pH 7.0, 1 mM
-mercaptoethanol, 100 µM NADH, 100 µM NADPH, 0.1 M LiCl, 30 µg of
fatty acid synthase extract from either strain UB1005 or RJH13 (fabI(Ts)) and, when added, 1 µg of purified His-tag FabI.
Cerulenin, when present, was at 100 µM. Reactions were
preincubated for 5 min at room temperature to allow the cerulenin to
inactivate the FabB and FabF condensing enzymes and then initiated by
the addition of 25 µM [2-
C]malonyl-CoA (specific activity, 57
Ci/mmol). After incubation at 37 °C for 10 min, reactions were
stopped by placing the tubes in an ice slurry. Gel loading buffer (10
µl) was then added, and 40 µl of the incubation mixture was
fractionated by conformationally sensitive gel electrophoresis in 13%
acrylamide gels containing 0.5 M urea(15) .
Electrophoresis was performed at 25 °C, 32 mA/gel. Fixed gels were
soaked in En
Hance (DuPont NEN) and then subjected to
fluorography at -80 °C.To investigate the reduction of
long-chain saturated and unsaturated acyl-ACPs, reactions were
performed as above, except that the acetyl-CoA was replaced with
tetradecanoyl-ACP or cis-7-tetradecenoyl-ACP, respectively.
Reactions were performed with and without 100 µM NADH and
1 µg of His-tag FabI. The products were analyzed by
conformationally sensitive gel electrophoresis in 2.5 M urea,
15% acrylamide gels to separate the long-chain products.
Reconstruction of Fatty Acid Synthesis in
Vitro
A single cycle of fatty acid elongation was
reconstructed in vitro by sequentially adding the individual
enzymes that catalyze each reaction in the cycle. The reaction mix
included, in a volume of 40 µl, 50 µM ACP, 1 mM
-mercaptoethanol, 0.1 M
Na
PO
, pH 7.0, 100 µM NADH, 100
µM NADPH, 100 µM acetyl-CoA, and 50
µM [
C]malonyl-CoA (specific
activity, 57 mCi/mmol). Purified, His-tagged enzymes were added at
0.2-0.6 µg/assay with FabD being added last to initiate the
reaction. Reactions were incubated at 37 °C for 20 min and then
stopped by placing into an ice slush. Analysis of the ACP thioesters
was performed by conformationally sensitive gel electrophoresis of the
products in 13% polyacrylamide gel containing 0.5 M urea, and
the bands were visualized by fluorography.
RESULTS AND DISCUSSION
Protein Purification and Substrate
Synthesis
We purified the five enzymes required to carry
out a single cycle of fatty acid synthesis to investigate the role of
each enzyme in completing the cycle. Each gene encoding a component of
the cycle was cloned into the pET-15b vector, and the proteins were
purified by affinity chromatography as described under
``Experimental Procedures.'' In all cases, active enzymes
were obtained in a highly purified state (Fig. 2). Acyl-ACPs
(14:0 and 14:1
7) were prepared using the acyl-ACP synthetase
method (12) and were pure (>95%) as judged by
conformationally sensitive gel electrophoresis (not shown).
Figure 2:
Purification of FabD, FabH, FabG, FabA,
and FabI proteins. The coding sequences for each of these proteins was
cloned into the pET-15b His-tag expression vector, expressed to high
levels following isopropyl-1-thio-
-D-galactopyranoside
induction, and purified by chromatography on
Ni
-agarose as described under ``Experimental
Procedures.'' The purity of each protein preparations used was
examined by SDS gel electrophoresis on 12% polyacrylamide
gels.
Reconstruction of Fatty Acid Biosynthesis with
Purified Components
A single round of fatty acid synthesis
was reconstructed in vitro by the sequential addition of
purified components to positively identify the intermediates formed by
each of the enzymes in the cycle and to verify the role of FabI in
completing rounds of elongation (Fig. 3). The addition of
malonyl-CoA:ACP transacylase plus
[2-
C]malonyl-CoA led to the formation of
[
C]malonyl-ACP. The addition of
-ketoacyl-ACP synthase III (FabH) resulted in the conversion of
malonyl-ACP to
-ketobutyryl-ACP. The gel electrophoresis technique
underestimated the actual amount of
-ketobutyryl-ACP formed
because the high pH, temperature, and urea used to achieve the
separation led to degradation and low recovery of the unstable
-ketoesters. The addition of
-ketoacyl-ACP reductase (FabG)
to the mixture (which also contained NADPH) resulted in the complete
conversion of the malonyl-ACP to
-hydroxybutyryl-ACP.
Significantly, the addition of
-hydroxyacyl-ACP dehydrase (FabA)
to the mixture resulted in only an 8% conversion of
-hydroxybutyryl-ACP to crotonyl-ACP. Increasing the amount of FabA
enzyme in the assay mixture did not lead to increased formation of
crotonyl-ACP (Fig. 3). The ratio of
-hydroxybutyryl-ACP and
crotonyl-ACP products was determined in this and several other
experiments by densitometric analysis of the fluorograph. Crotonyl-ACP
accounted for 8-11% of the total density in the
-hydroxybutyryl-ACP plus crotonyl-ACP bands and was independent of
the amount of FabA enzyme added to the assay or the length of the
incubation. These data indicated that the FabA reaction rapidly reached
an equilibrium that favored
-hydroxybutyryl-ACP. The addition of
FabI to the reaction mixture yielded butyryl-ACP, and the extent of
butyryl-ACP formation increased as the amount of FabI in the assay
increased. These data show that the activity of FabI is responsible for
pulling cycles of fatty acid elongation.
Figure 3:
Reconstitution of a cycle of fatty acid
synthesis with purified enzymes. A single cycle of fatty acid
biosynthesis was reconstructed in vitro by the sequential
addition of each purified enzyme to a reaction mixture containing
NADPH, NADH, and ACP as cofactors and acetyl-CoA and
[2-
C]malonyl-CoA as substrates as described
under ``Experimental Procedures.'' The products were analyzed
by conformationally sensitive gel electrophoresis using 13%
polyacrylamide gels containing 0.5 M urea.
Correction of the Fatty Acid Synthesis Defect in
Extracts of fabI(Ts) Mutants with Purified FabI
Extracts
isolated from wild-type and fabI(Ts) defective strains were
used to examine in vitro the role of the FabI protein in fatty
acid elongation (Fig. 4). As previously reported(20) ,
extracts from the wild-type strain incorporated
[
C]malonyl-CoA into butyryl-ACP and long-chain
acyl-ACP. The addition of cerulenin blocked the activity of FabB and
FabF but not FabH, leading to the accumulation of butyryl-ACP. Extracts
from strain RJH13 (fabI(Ts)) were unable to form either
butyryl-ACP or long-chain acyl-ACP either in the presence or the
absence of cerulenin. Two intermediates accumulated in these reactions.
The major intermediate comigrated with
-hydroxybutyryl-ACP, and
the minor species comigrated with crotonyl-ACP. Densiometric analysis
of the fluorograph showed that the crotonyl-ACP band comprised 10% of
the total density of the
-hydroxybutyryl-ACP plus crotonyl-ACP
bands. The ratio of these two products formed in the crude cell extract
was virtually identical to the ratio of products observed in the
purified system in the absence of FabI (Fig. 3). The addition of
purified FabI protein to the extract from the fabI(Ts) strain
restored the ability of the extract to synthesize both long-chain
acyl-ACP and butyryl-ACP in the presence of cerulenin. These data show
that FabI is essential for the first cycle of fatty acid elongation and
is the only component missing from the extracts of strain RJH13 (fabI(Ts)).
Figure 4:
Restoration of butyryl-ACP and long-chain
acyl-ACP formation in extracts of fabI(Ts) by the addition of
purified FabI protein. Cell-free extracts of strain UB1005 (wild type)
and RJH13 (fabI(Ts)) were prepared, and fatty acid synthase
reactions containing NADPH, NADH, ACP, acetyl-CoA, and
[2-
C]malonyl-CoA were performed as described
under ``Experimental Procedures.'' Cerulenin (100
µM) was added to the indicated reactions (+) to block
the activity of
-ketoacyl-ACP synthases I and II (FabB and FabF).
Purified His-tag FabI (1 µg) was added to the indicated assays. The
products were analyzed by conformationally sensitive gel
electrophoresis using 13% polyacrylamide gels containing 0.5 M urea, and the bands were visualized by
fluorography.
The data suggested that FabI participated in
subsequent elongation cycles, but the possibility remained that another
enoyl-ACP reductase (the NADPH-dependent enzyme predicted by Weeks and
Wakil (5) ) was present in the extracts and contributed to the
elongation of saturated or unsaturated fatty acids to produce
long-chain acyl-ACP (Fig. 4). To verify that FabI was capable of
participating in the elongation of these long-chain species and to rule
out the presence of another enzyme, we examined the elongation of
14:0-ACP (
)and 14:1
7-ACP (Fig. 5). Extracts from
strain RJH13 (fabI(Ts)) were unable to convert either 14:0-ACP
or 14:1
7-ACP to 16:0-ACP or 16:1
9-ACP, respectively. These
extracts contained FabB (or FabF) that catalyzed the condensation of
[
C]malonyl-CoA with the acyl-ACP, FabG, and FabZ
(or FabA) that carried out the NADPH-dependent reduction of the
-ketoacyl-ACPs and the dehydration of
-hydroxyacyl-ACP to the trans-2-enoyl-ACP intermediate. Two intermediates accumulated
in each experiment that corresponded in electrophoretic mobility to the
-hydroxyacyl and trans-2-enoyl intermediates in the
elongation cycles. Like the electrophoretic relationship between
-hydroxybutyryl-ACP and crotonyl-ACP observed in Fig. 3,
the long-chain
-hydroxyacyl intermediates migrated more slowly
than the corresponding acyl-ACP, and the long-chain trans-2-enoyl intermediates migrated slightly more quickly
than the corresponding acyl-ACP. These relative mobilities were the
same as reported previously for long-chain
-hydroxyacyl and trans-2-enoyl acyl-ACP(4) . The ratio of the
long-chain
-hydroxyacyl-ACP to trans-2-enoyl-ACP was
quantitated by densitometry for each experiment (Fig. 5). The
average ratio of these two intermediates was 1:1 indicating that the
equilibrium for the dehydrase operating on the long-chain
-hydroxyacyl-ACPs was more favorable for the formation of the trans-2-enoyl-ACP product than was observed with the
short-chain intermediates ( Fig. 3and Fig. 4). The
addition of purified FabI to these extracts restored the ability of the
extracts to elongate 14:0-ACP to 16:0-ACP and 14:1
7-ACP to
16:1
9-ACP. NADH was required in addition to FabI for the formation
of acyl-ACP. These data rule out the existence of a NADPH-dependent
enoyl-ACP reductase participating in the elongation of either
long-chain saturated or unsaturated fatty acids. These experiments also
verified that FabI is capable of reducing the trans-2 double
bond in both long-chain saturated and unsaturated enoyl-ACP.
Figure 5:
Purified FabI protein restores the
elongation of both saturated and unsaturated long-chain acyl-ACP in
extracts from fabI(Ts) mutants. The elongation of either
14:0-ACP (A) or 14:1
7-ACP (B) was studied in
extracts from strain RJH13 (fabI(Ts)) in incubations
containing NADPH and [2-
C]malonyl-CoA either in
the presence or the absence of NADH were performed as described under
``Experimental Procedures.'' Purified His-tag FabI (1 µg)
was added to the indicated assays. Products were separated by
conformationally sensitive gel electrophoresis on 15% polyacrylamide
gels containing 2.5 M urea, and the bands were visualized by
fluorography.
Composition of the ACP Pool in fabI(Ts)
Mutants
Previously, we concluded from the analysis of fatty
acid synthesis and long-chain acyl-ACP and malonyl-CoA accumulation in fabI(Ts) mutants that FabI was required for the first
elongation cycle that forms butyryl-ACP(16) . We also noted a
change in the composition of the short-chain ACP thioester pool, and we
examined this pool more closely to determine if our in vitro experiments with extracts from fabI(Ts) mutants and
purified components correlated with the in vivo results.
Strain RJH13 (panD2 fabI(Ts)) was labeled with
-[
H]alanine to uniformly label the ACP pool,
and the composition of this pool was analyzed by conformationally
sensitive gel electrophoresis at both the permissive and nonpermissive
temperatures using the wild-type strain SJ16 for comparison (Fig. 6). To facilitate the analysis, a portion of each culture
was incubated with cerulenin, an irreversible inhibitor of the FabB and
FabF condensing enzymes. At the permissive temperature (30 °C), the
major ACP species in strain RJH13 co-migrated with acetyl-ACP with
significant amounts of malonyl-ACP also detected. This pattern of ACP
intermediates was essentially the same as in the control strain SJ16.
The addition of cerulenin to strain RJH13 grown at 30 °C led to the
accumulation of butyryl-ACP, which is the same major change in the pool
composition found in the wild-type control strain SJ16. In contrast,
treatment of strain RJH13 with cerulenin after a temperature shift to
42 °C did not lead to the accumulation of butyryl-ACP indicating
that the first cycle of fatty acid biosynthesis was blocked in the fabI(Ts) mutants. Shifting strain RJH13 to the nonpermissive
temperature (42 °C) lead to a significant change in the ACP pool
composition. Acetyl-ACP essentially disappeared, malonyl-ACP
accumulated, and a new species migrating slightly more quickly than ACP
appeared. We also observed the appearance of a new minor species that
migrated just ahead of butyryl-ACP. The migration positions of these
two ACP intermediates in vivo corresponded to the migration
positions of
-hydroxybutyryl-ACP and crotonyl-ACP ( Fig. 3and Fig. 4). One important conclusion from these
experiments was that the ACP pool composition of fabI(Ts)
mutants at the nonpermissive temperature (Fig. 6) was
essentially identical to the distribution of products observed in the in vitro system with either purified components (Fig. 3) or extracts from fabI(Ts) mutants (Fig. 4). Densitometric analysis of the fluorograph showed that
the band with the migration position of crotonyl-ACP was 13% of the
total density in the
-hydroxybutyryl-ACP plus crotonyl-ACP bands.
Thus, our in vitro reconstitution experiments accurately
reflect the properties of the fatty acid synthase in vivo.
Figure 6:
Composition of the ACP pool in fabI(Ts) mutants. Strain RJH13 (panD fabI(Ts)) was
grown at 30 °C and strain SJ16 (panD) at 37 °C with
-[3-
H]alanine to a density of 5
10
cells/ml. The strain RJH13 culture was split, and one
half was shifted to 42 °C. 10 min later, samples were removed,
remaining cultures were treated with 1 mg/ml cerulenin for a futher 10
min, and additional 1-ml samples were removed. A portion of the strain
SJ16 culture was also treated with cerulenin for 10 min. The ACP pool
composition was analyzed by conformationally sensitive gel
electrophoresis in 13% polyacrylamide gels containing 0.5 M urea, and the bands were visualized by fluorography as described
under ``Experimental Procedures.'' The migration position of
acyl-ACP standards are indicated to the right.
Conclusions
Our results point to
enoyl-ACP reductase (the fabI gene product) as a determinant
factor in completing rounds of fatty acid elongation (Fig. 1).
The reactions catalyzed by FabH, FabG, and FabI all result in the
extensive conversion to the next intermediate in the cycle as depicted
by the thickness of the arrows in Fig. 1. The amount of
butyryl-ACP formed is dependent on the amount of FabI protein added due
to the dehydration reaction reaching a rapid equilibrium that favors
the accumulation of
-hydroxybutyryl-ACP over crotonyl-ACP by a
ratio of 9:1. These data indicate that increasing the rate of
initiation of a cycle of fatty acid elongation by a factor of 10 would
only double the amount of enoyl-ACP product produced by FabA. Although
it is not clear whether FabA, FabZ, or both contribute to the
dehydration of
-hydroxybutyryl-ACP in vivo, the fact that
our in vitro reconstitution experiments reflect the properties
of the fatty acid synthase in vivo provides compelling support
for the conclusion that FabI activity plays a determinant role in
completing rounds of fatty acid elongation.Our experiments show
that enoyl-ACP reductase (the fabI gene product) is the only
reductase required to complete both saturated and unsaturated fatty
acid synthesis in the type II, dissociated fatty acid synthase system
of E. coli. Extracts derived from fabI(Ts) strains
were unable to synthesize butyryl-ACP or complete the elongation of
long-chain saturated or unsaturated fatty acids without supplementation
with exogenous FabI. We found no evidence for the NADPH-dependent
isoform postulated by Weeks and Wakil(5) . The Mycobacterium tuberculosis FabI analog (inhA) is also
specific for NADH (23) and participates in ACP-dependent fatty
acid synthesis in a type II system similar to that in M.
smegmatis(24) . We do not have a complete picture of the
substrate specificity of InhA enoyl-ACP reductase, but the enzyme
effectively reduces trans-2-octenoyl-ACP as well as long-chain
intermediates (24) indicating a broad chain length
specificity.
Hypothesis
The fact that a single
enoyl-ACP reductase plays a determinant role in completing cycles of
chain elongation suggests that FabI may be a key regulatory component
in dissociated fatty acid synthase systems. Two aspects of fatty acid
synthesis may be influenced by modulation of FabI activity. First, FabI
may participate in establishing the basal saturated:unsaturated fatty
acid ratio. FabA is the critical branch point because it is the only
dehydrase capable of forming both cis-3-decenoyl-ACP and trans-2-decenoyl-ACP(1) . FabB is essential for
unsaturated fatty acid synthesis presumably because it is the only
condensing enzyme capable of utilizing cis-3-decenoyl-ACP. In
contrast, FabI reduces trans-2-decanoyl-ACP to decanoyl-ACP to
initiate saturated fatty acid biosynthesis. Genetic manipulation of the
intracellular levels of FabB and FabA establish that altering the
relative ratios of these two proteins has a dramatic impact on the
saturated:unsaturated fatty acid ratio(25) . One testable
hypothesis is that increased levels of FabI protein would increase
synthesis of saturated fatty acids by increasing the utilization of trans-2-decenoyl-ACP. Second, FabI may be a target for
regulation of the total rate of chain elongation. Acyl-ACPs are
emerging as key regulators of the rate of fatty acid biosynthesis, and
their destruction in vivo by the expression of acyl-ACP
thioesterases of bacterial (26, 27) or plant (28, 29, 30) origin leads to the acceleration
of fatty acid biosynthesis and the copious secretion of fatty acids
into the medium. Furthermore, down-regulation of fatty acid synthesis
following the cessation of phospholipid production at the
glycerol-phosphate acyltransferase step correlates with the elevation
of intracellular acyl-ACP(16, 31) . The intracellular
targets for acyl-ACP regulation are unknown. One tenable hypothesis is
that acyl-ACPs attenuate FabI activity by product inhibition, and this
mechanism may contribute to the regulation of pathway activity by
acyl-ACP.