©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of -Ketoacyl-Acyl Carrier Protein Synthase III (FabH) by Acyl-Acyl Carrier Protein in Escherichia coli(*)

(Received for publication, December 11, 1995; and in revised form, February 5, 1996)

Richard J. Heath (1) Charles O. Rock (1) (2)(§)

From the  (1)Department of Biochemistry, St Jude Children's Research Hospital, Memphis, Tennessee 38101 and the (2)Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

beta-Ketoacyl-acyl carrier protein (ACP) synthase III (the fabH gene product) condenses acetyl-CoA with malonyl-ACP to initiate fatty acid biosynthesis in the dissociated, type II fatty acid synthase systems typified by Escherichia coli. The accumulation of malonyl-acyl carrier protein (ACP) following the inhibition of a reconstituted fatty acid synthase system by acyl-ACP implicated synthase III (FabH) as a target for acyl-ACP regulation (Heath, R. J., and Rock, C. O.(1996) J. Biol. Chem. 271, 1833-1836); therefore, the FabH protein was purified and its biochemical and regulatory properties examined. FabH exhibited a K of 40 µM for acetyl-CoA and 5 µM for malonyl-ACP. FabH also accepted other acyl-CoAs as primers with the rank order of activity being acetyl-CoA approx propionyl-CoA butyryl-CoA. FabH utilized neither hexanoyl-CoA nor octanoyl-CoA. Acyl-ACPs suppressed FabH activity, and their potency increased with increasing acyl chain length between 12 and 20 carbon atoms. Nonesterified ACP was not an inhibitor. Acyl-ACP inhibition kinetics were mixed with respect to acetyl-CoA, but were competitive with malonyl-ACP, indicating that acyl-ACPs decrease FabH activity by binding to either the free enzyme or the acyl-enzyme intermediate. These data support the concept that the inhibition of chain initiation at the beta-ketoacyl-ACP synthase III step contributes to the attenuation of fatty acid biosynthesis by acyl-ACP.


INTRODUCTION

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 (^1)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.

beta-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) . beta-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) (^2)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-^14C]malonyl-CoA (specific activity, 57 mCi/mmol) and [1-^14C]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 beta-mercaptoethanol, 65 µM malonyl-CoA, 45 µM [1-^14C]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, beta-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 beta-mercaptoethanol, 0.1 M sodium phosphate, pH 7.0, 45 µM [2-^14C]malonyl-CoA (specific activity 57 mCi/mmol). The ACP was reduced by incubation in the buffer plus beta-mercaptoethanol for 30 min at 37 °C prior to addition to the assay tube, and the reaction was initiated by the addition of [2-^14C]malonyl-CoA. The amount of [2-^14C]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-beta-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(2) to a density of approximately 5 times 10^8 cells/ml. Isopropyl-1-thio-beta-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 beta-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 beta-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 beta-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 [^14C]acetyl-CoA (specific activity, 27 mCi/mmol), 0.1 M sodium phosphate, pH 7.0, 1 mM beta-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 [^14C]acetyl-CoA (specific activity, 27 mCi/mmol), 0.1 M sodium phosphate, pH 7.0, 100 µM ACP, 1 mM beta-mercaptoethanol, and 0.2-2.4 µg of enzyme. Reactions were incubated at 37 °C for 12 min, and the amount of [^14C]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 beta-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 beta-mercaptoethanol, 50 µM ACP, 100 µM NADPH, and the reactions initiated by the addition of 25 µM [2-^14C]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 beta-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-^14C]Acetyl-CoA was the other substrate. Long-chain acyl-ACPs (16:0- or 18:1Delta11-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-^14C]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(m) 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 (approx2 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 ^14C-labeled acyl-CoAs for each chain length; therefore, the FabH assay was modified to label the products with [2-^14C]malonyl-CoA and quantitate product formation by conformationally sensitive gel electrophoresis. Because beta-ketoacyl-ACP thioesters are not stable to the electrophoretic conditions employed, we also included beta-ketoacyl-ACP reductase (FabG) and NADPH to the reactions to convert the unstable beta-ketoacyl-ACPs to their corresponding stable beta-hydroxyacyl-ACPs for analysis. As anticipated, this system efficiently converted acetyl-CoA to beta-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-^14C]malonyl-ACP as described under ``Experimental Procedures.'' The reaction mixtures were separated by conformationally sensitive gel electrophoresis and the formation of the beta-hydroxyacyl-ACPs was determined by autoradiography. Abbreviations are: Mal-ACP, malonyl-ACP; Ac-ACP, acetyl-ACP; beta-OH 4:0-ACP, beta-hydroxybutyryl-ACP; beta-OH 5:0-ACP, beta-hydroxypentanoyl-ACP; beta-OH 6:0-ACP, beta-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 beta-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 [^14C]malonyl-ACP was used as the substrate in these assays. Similar experiments performed with [^14C]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 [^14C]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 18:1Delta11-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(2). 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 beta-[3-^3H]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 times 10^8 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 beta-[3-^3H]alanine (0.5 µM, specific activity 91 Ci/mmol). At a density of 5 times 10^8 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 beta-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 20:1Delta13-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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM34496, Cancer Center (CORE) Support Grant CA 21765, and the American and Lebanese Syrian Associated Charities. 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: Dept. of Biochemistry, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38101. Tel.: 901-495-3491; Fax: 901-525-8025; charles.rock{at}stjude.org.

(^1)
The abbreviations used are: ACP, acyl carrier protein; acyl-ACP, acyl-acyl carrier protein; glycerol-P, sn-glycerol-3-phosphate; ppGpp, guanosine-3`-diphosphate-5`-diphosphate; 18:1Delta11 (number of carbon atoms:number of double bonds), position of the double bond.

(^2)
Lowercase italic type (fabH) denotes a discrete gene, and uppercase Roman type (FabH) signifies the protein product of the gene.


ACKNOWLEDGEMENTS

We thank Suzanne Jackowski for informative discussions and R. Brent Calder for expert technical assistance.


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