The multifunctional polypeptide chain of rabbit mammary fatty acid synthase contains a domain homologous with the acyl carrier protein of Escherichia coli

The phosphopantetheine thiol of rabbit mammary fatty acid synthase was specifically alkylated using chloro[14C]acetyl-CoA and a radioactive fragment generated by limited elastase digestion of the modified protein was purified by gel filtration. We have previously mapped this fragment to an internal location in the 250 000-Mr polypeptide adjacent to the thioesterase domain [Eur. J. Biochem. 130, 185-193 (1983)]. The purified fragment had apparent molecular weights of 23 000 by gel filtration and 10 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, while amino acid analysis indicated a minimal molecular weight of 10 400. We have determined the amino acid sequence of the first 64 residues of the fragment. The phosphopantetheine moiety is esterified to a serine at residue 38 in the sequence. When the sequences of the rabbit acyl carrier fragment and the 8847-Mr acyl carrier protein of Escherichia coli are aligned, 17 out of 64 residues are identical. These results suggest that the limited proteolysis delineates an internal acyl carrier domain within the rabbit protein and provide the first clear evidence that multifunctional fatty acid synthases have arisen by fusion of ancestral monofunctional proteins.     

Specific modification of the condensation domain of fatty acid synthase and the determination of the primary structure of the modified active site peptides

Fatty acid synthase from the uropygial gland of goose was inactivated by iodoacetamide with a second-order rate constant of 1.3 M-1 S-1 at pH 6.0 and 25 degrees C. Of the seven component activities of the synthase, only the condensation activity was significantly inhibited by iodoacetamide modification. Since preincubation of the enzyme with acetyl-CoA, but not with malonyl-CoA, protected the enzyme from inactivation by iodoacetamide, it is suggested that iodoacetamide probably modified the primer-binding thiol group at the condensation active site. Determination of the stoichiometry of modification was done using [1-14C]iodoacetamide that was purified by high-performance liquid chromatography. Graphical analysis of the data showed that binding of 1.2 carboxamidomethyl groups per subunit of fatty acid synthase would result in complete inhibition of the enzyme activity, suggesting that there is one condensation domain per subunit of fatty acid synthase. Analysis of the tryptic peptide map of the enzyme that was modified with [1-14C]iodoacetamide in the presence and absence of acetyl-CoA revealed that acetyl-CoA prevented the labeling of a major radioactive peptide and a minor radioactive peptide. These two peptides were purified by high-performance liquid chromatography. Amino acid analysis of these two peptides revealed that the major radioactive peptide contained S-carboxymethylcysteine while the minor radioactive peptide did not. However, the latter peptide contained beta-alanine, suggesting that this peptide was from the acyl carrier protein segment of fatty acid synthase and that the iodoacetamide treatment resulted in modification of the pantetheine thiol, although to a lower extent than the primer-binding thiol. The sequence of the primer-binding active site peptide from the condensation domain was H2N-Gly-Pro-Ser-Leu-Ser-Ile-Asp- Thr-Ala-Cys(carboxamidomethyl)-X-Ser-Ser-Leu-Met-Ala-Leu-Glu-Asn-A la-Tyr-Lys- COOH, the first reported sequence of the condensation active site from a vertebrate fatty acid synthase. The acyl carrier protein segment showed extensive sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the phosphopantetheine attachment, and the sequence was H2N-Asp-Val-Ser-Ser-Leu- Asn-Ala-Asp-Ser-Thr-Leu-Ala-Asp-Leu-Gly-Leu-Asp-Ser(4'-phosphopanteth ein e) -Leu-Met-Gly-Val-Glu-Val-Arg-COOH.     

Characterization of the fatty acid synthetase system of Curtobacterium pusillum

Curtobacterium pusillum contains 11-cyclohexylundecanoic acid as a major component of cellular fatty acids. A trace amount of 13-cyclohexyltridecanoic acid is also present. Fatty acids other than omega-cyclohexyl fatty acids present are 13-methyltetradecanoic, 12-methyltetradecanoic, n-pentadecanoic, 14-methylpentadecanoic, 13-methylpentadecanoic, n-hexadecanoic, 15-methylhexadecanoic, 14-methylhexadecanoic, and n-heptadecanoic acids. The fatty acid synthetase system of this bacterium was studied. Various 14C-labeled precursors were added to the growth medium and the incorporation of radioactivity into cellular fatty acids was analyzed. Sodium [14C]acetate and [14C]glucose were incorporated into almost all species of cellular fatty acids, the incorporation into 11-cyclohexylundecanoic acid being predominant. [14C]Isoleucine was incorporated into 12-methyltetradecanoic and 14-methylhexadecanoic acids: [14C]leucine into 13-methyltetradecanoic and 15-methylhexadecanoic acids; and [14C]valine into 14-methylpentadecanoic acid. [14C]-Shikimic acid was incorporated almost exclusively into omega-cyclohexyl fatty acids. The fatty acid synthetase activity of the crude enzyme preparation of C. pusillum was reconstituted on the addition of acyl carrier protein. This synthetase system required NADPH and preferentially utilized cyclohexanecarbonyl-CoA as a primer. The system was also able to use branched- and straight-chain acyl-CoAs with 4 to 6 carbon atoms effectively as primers but was unable to use acetyl-CoA. However, if acetyl acyl carrier protein was used as the priming substrate, the system produced straight-chain fatty acids. The results imply that the specificity of the initial acyl-CoA:acyl carrier protein acyltransferase dictates the structure of fatty acids synthesized and that the enzymes catalyzing the subsequent chain-elongation reactions do not have the same specificity restriction.     

Active site directed inactivation of rat mammary gland fatty acid synthase by 3-chloropropionyl coenzyme A

3-Chloropropionyl coenzyme A (CoA) irreversibly inhibits rat mammary gland fatty acid synthase. Enzyme inactivation proceeds with first-order kinetics. NADPH (150 microM) as well as acetyl-CoA (500 microM) affords protection against inactivation, suggesting that the inhibitor is active site directed. In contrast, malonyl-CoA (500 microM) offers little protection. With chloro [1-14C]propionyl-CoA, stoichiometries of modification that approach one per enzyme protomer (240 kilodaltons) have been measured. When chloropropionyl-[3'-32P]CoA is used for inactivation, modification stoichiometries are less than 10% of the value observed in the 14C labeling experiments, suggesting that acylation of the enzyme occurs. Radioactivity remains associated with the 14C-labeled protein after performic acid oxidation, indicating that another linkage, in addition to the thio ester adduct, is formed during inactivation. Recovery of [( 14C]carboxyethyl)cysteine from digests of the inactivated enzyme indicates that alkylation of an active site cysteine occurs. The cysteamine sulfhydryl of the acyl carrier peptide is clearly not the site of modification. Loss of overall enzyme activity is tightly linked to decreases in the ketoacyl synthase partial reaction. This observation, coupled with the differential protection measured with acetyl-CoA and malonyl-CoA, suggests that the reagent modifies a residue at the active site involved in condensation. While inactivated enzyme shows good ketoacyl reductase activity when S-(acetoacetyl)-N-acetylcysteamine is used as a substrate, only poor activity for this partial reaction is measured when acetoacetyl-CoA is the substrate. This implies that the function of the acyl carrier peptide (ACP) is impaired during the inactivation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.     

Evidence that the multifunctional polypeptides of vertebrate and fungal fatty acid synthases have arisen by independent gene fusion events

The enoyl reductase (NADPH binding site) of rabbit mammary fatty acid synthase has been radioactively labelled using pyridoxal phosphate and sodium [3H]borohydride. Using this method we have been able to add this site to the four sites whose location has already been mapped within the multifunctional polypeptide chain of the protein. The results show that the enoyl reductase lies between the 3-oxoacylsynthase and the acyl carrier. This confirms that the active sites occur in a different order on the single multifunctional polypeptide of vertebrate fatty acid synthase and the two multifunctional polypeptides of fungal fatty acid synthase, and suggests that these two systems have arisen by independent gene fusion events.     

The isolation of acyl carrier protein from the pigeon liver fatty acid synthetase complex1 II

A low molecular weight protein of less than 10, 000 Daltons has been isolated from Subunit I (β-ketoacyl thioester reductase) of the pigeon liver fatty acid synthetase complex and purified to homogeneity. This protein contains all of the [¹⁴C]-labeled pantetheine incorporated into the fatty acid synthetase on injection of [¹⁴C]-labeled pantetheine into pigeons. It also has one β-alanine and one sulfhydryl group. This protein is an acceptor of an acetyl group from acetyl-CoA and a malonyl group from malonyl-CoA in the presence of Subunit II (transacylase). In these respects it is very similar to $\text{E. coli}$ acyl carrier protein.     

Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase.

Goat mammary fatty acid synthetase was labelled in the acyltransferase domain by formation of O-ester intermediates by incubation with [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Tryptic-digest and CNBr-cleavage peptides were isolated and purified by high-performance reverse-phase and ion-exchange liquid chromatography. The sequences of the malonyl- and acetyl-labelled peptides were shown to be identical. The results confirm the hypothesis that both acetyl and malonyl groups are transferred to the mammalian fatty acid synthetase complex by the same transferase. The sequence is compared with those of other fatty acid synthetase transferases.     

An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A

1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3'-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ;burst' release of CoA proportional in amount to the quantity of enzyme present. The ;burst' of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-(14)C]acetyl-CoA yielded protein-bound acetyl groups. The K(eq.) for the acetylation was 1.2 at pH7.0 and 4 degrees C. Acetyl-labelled synthase was isolated free from [1-(14)C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-(14)C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-(14)C]acetyl-CoA or 3-hydroxy-3-methyl-[(14)C]glutaryl-CoA respectively. 4. The isolated [1-(14)C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4 degrees C with a first-order rate constant of 0.005min(-1). This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.     

Mitochondrial fatty acid synthesis in Trypanosoma brucei

Whereas other organisms utilize type I or type II synthases to make fatty acids, trypanosomatid parasites such as Trypanosoma brucei are unique in their use of a microsomal elongase pathway (ELO) for de novo fatty acid synthesis (FAS). Because of the unusual lipid metabolism of the trypanosome, it was important to study a second FAS pathway predicted by the genome to be a type II synthase. We localized this pathway to the mitochondrion, and RNA interference (RNAi) or genomic deletion of acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase indicated that this pathway is likely essential for bloodstream and procyclic life cycle stages of the parasite. In vitro assays show that the largest major fatty acid product of the pathway is C16, whereas the ELO pathway, utilizing ELOs 1, 2, and 3, synthesizes up to C18. To demonstrate mitochondrial FAS in vivo, we radio-labeled fatty acids in cultured procyclic parasites with [(14)C]pyruvate or [(14)C]threonine, either of which is catabolized to [(14)C]acetyl-CoA in the mitochondrion. Although some of the [(14)C]acetyl-CoA may be utilized by the ELO pathway, a striking reduction in radiolabeled fatty acids following ACP RNAi confirmed that it is also consumed by mitochondrial FAS. ACP depletion by RNAi or gene knockout also reduces lipoic acid levels and drastically decreases protein lipoylation. Thus, octanoate (C8), the precursor for lipoic acid synthesis, must also be a product of mitochondrial FAS. Trypanosomes employ two FAS systems: the unconventional ELO pathway that synthesizes bulk fatty acids and a mitochondrial pathway that synthesizes specialized fatty acids that are likely utilized intramitochondrially.     

Cerulenin resistance in a cerulenin-producing fungus. III. Studies on active-site peptides of fatty acid synthetase from Cephalosporium caerulens

Active-site peptides of acetyl transferase, condensing enzyme and acyl carrier protein in the neighborhood of the prosthetic group, 4'-phosphopantetheine, of Cephalosporium caerulens fatty acid synthetase were investigated. The enzyme was reacted with [14C]acetyl-CoA or [14C]iodoacetamide. 14C-Labeled enzyme was digested with pepsin, trypsin or both. 14C-Labeled peptides were isolated by several purification procedures. The amino acid sequence of the active site of condensing enzyme was determined to be Tyr-Gln-Val-Glu-Ser-Cys-Pro-Ile-Leu-Glu-Gly-Lys and that of acetyl transferase was Phe-Ser-Gly-Ala-Thr-Gly-His-Ser-Gln-Gly. The amino acid composition around the 4'-phosphopantetheine-carrying serine was determined to be Asx2, Thr, Ser, Glx3, Gly2, Ala, Ile, Leu3, and Lys. When these active-site peptides were compared with those of Saccharomyces cerevisiae synthetase, a high degree of homology was observed in the active-site peptides of the acetyl transferase and acyl carrier protein domains. However, that of the condensing enzyme domain gave lower homology. These findings may support the assumption that the low reactivity of cerulenin with C. caerulens synthetase is a consequence of the structure of the condensing enzyme domain.     

Identification and molecular characterization of the beta-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, beta-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.     

Structure of the mitochondrial beta-ketoacyl-[acyl carrier protein] synthase from Arabidopsis and its role in fatty acid synthesis

Mitochondrial fatty acid synthesis is catalyzed by a dissociated fatty acid synthase similar to those of plant plastids and bacteria. The crystal structure of a mitochondrial beta-ketoacyl-[acyl carrier protein] synthase (mtKAS), namely that from Arabidopsis thaliana, has been determined for the first time. This enzyme accomplishes the vital condensation steps in constructing fatty acid carbon skeletons. The product profile of mtKAS is unusual in that C8 and C(14-16) fatty acyl chains predominate. An enzyme architecture that likely is the basis for the observed bimodal profile of mtKAS products can be derived from the shape of the acyl binding pocket.     

3-Hydroxy-3-methylgutaryl-CoA synthase. Participation of acetyl-S-enzyme and enzyme-S-hydroxymethylgutaryl-SCoA intermediates in the reaction.

Acetyl-CoA reacts stoichiometrically with a cysteinyl sufhydryl group of avian liver 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase to yield acetyl-S-enzyme (Miziorko H.M., Clinkenbeard, K.D., Reed, W.D., and Lane, M.D. (1975) J. Biol. Chem. 250, 5768-5773). Evidence that acetyl-S-enzyme condenses with the second substrate, acetoacetyl CoA, to form enzyme-S-HMG-SCoA has been obtained by trapping and characterizing this putative intermediate. [14C]Acetyl-S-enzyme was incubated briefly at -25 degrees with acetoacetyl-CoA, precipitated with trichloroacetic acid, and the labeled acylated enzyme species were isolated. Performic acid oxidation of the precipitated [14C]acyl-S-enzyme intermediates produced volatile [14C]acetic acid from unreacted [14C]acetyl-S-enzyme and nonvolatile [14C]3-hydroxy-3-methyl glutaric acid from enzyme-S-[14C]HMG-SCoA. Condensation of unlabeled acetyl-S-enzyme with [14C]aceto-acetyl-CoA or acetoacetyl-[3H]CoA also produced labeled enzyme-S-HMG-SCoA. Thus, the acetyl moiety from acetyl-CoA and the acetoacetyl and CoA moieties from acetoacetyl-CoA all are incorporated into the HMG-CoA which is covalently-linked to the enzyme. Enzyme-S-[14C]HMG-SCoA was subjected to proteolytic digestion under conditions favorable for intramolecular S to N acyl transfer in the predicted cysteine-S-[14C]HMG-SCoA fragment. Performic acid oxidation of the protease-digested material yields N-[14C]HMG-cysteic acid indicating that HMG-CoA had been covalently bound to the enzyme via the -SH of an active site cysteine. An isotope trapping technique was employed to test the kinetic competence of acetyl-S-enzyme as an intermediate in the HMG-CoA synthase-catalyzed reaction. Evidence is presented which indicates that the rate of condensation of acetoacetyl-CoA with acetyl-S-enzyme to form enzyme-S-HMG-SCoA is more rapid than either the acetylation of the synthase by acetyl-CoA or the overall forward reaction leading to HMG-CoA. These observations, together with indirect evidence that hydrolysis of enzyme-S-HMG-SCoA is extremely rapid, suggest that acetylation of synthase is the rate-limiting step in HMG-CoA synthesis.     

Evidence for an Elongation/Reduction/C1-Elimination Pathway in the Biosynthesis of n-Heptane in Xylem of Jeffrey Pine

The biosynthetic pathway to n-heptane was investigated by examining the effect of the [beta]-keto acyl-acyl carrier protein synthase inhibitor (2R,3S)-2,3-epoxy-4-oxo-7E,10E-dodecadienamide (cerulenin), a thiol reagent ([beta]-mercaptoethanol), and an aldehydetrapping reagent (hydroxylamine) on the biosynthesis of n-[14C]heptane and putative intermediates in xylem sections of Jeffrey pine (Pinus jeffreyi Grev.& Balf.) incubated with [14C]acetate. Cerulenin inhibited C18 fatty acid biosynthesis but had relatively little effect on radiolabel incorporation into C8 fatty acyl groups and n-heptane. [beta]-Mercaptoethanol inhibited n-heptane biosynthesis, with a corresponding accumulation of radiolabel into both octanal and 1-octanol, whereas hydroxylamine inhibited both n-heptane and 1-octanol biosynthesis, with radiolabel accumulation in octyl oximes. [14C]Octanal was converted to both n-heptane and 1-octanol when incubated with xylem sections, whereas [14C]1-octanol was converted to octanal and n-heptane in a hydroxylamine-sensitive reaction. These results suggest a pathway for the biosynthesis of n-heptane whereby acetate is polymerized via a typical fatty acid synthase reaction sequence to yield a C8 thioester, which subsequently undergoes a two-electron reduction to generate a free thiol and octanal, the latter of which alternately undergoes an additional, reversible reduction to form 1-octanol or loss of C1 to generate n-heptane.     

The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids.

1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo.     

Mechanistic studies on beta-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes

Thiolase proceeds via covalent catalysis involving an acetyl-S-enzyme. The active-site thiol nucleophile is identified as Cys89 by acetylation with [14C]acetyl-CoA, rapid denaturation, tryptic digestion, and sequencing of the labeled peptide. The native acetyl enzyme is labile to hydrolytic decomposition with t 1/2 of 2 min at pH 7, 25 degrees C. Cys89 has been converted to the alternate nucleophile Ser89 by mutagenesis and the C89S enzyme overproduced, purified, and assessed for activity. The Ser89 enzyme retains 1% of the Vmax of the Cys89 enzyme in the direction of acetoacetyl-CoA thiolytic cleavage and 0.05% of the Vmax in the condensation of two acetyl-CoA molecules. A covalent acetyl-O-enzyme intermediate is detected on incubation with [14C]acetyl-CoA and isolation of the labeled Ser89-containing tryptic peptide. Comparisons of the Cys89 and Ser89 enzymes have been made for kinetic and thermodynamic stability of the acetyl enzyme intermediates both by isolation and by analysis of [32P]CoASH/acetyl-CoA partial reactions and for rate-limiting steps in catalysis with trideuterioacetyl-CoA.     

Reaction of chloroacetyl-CoA with rabbit fatty acid synthase: A new method to label specifically and quantify pantetheine prosthetic groups

The substrate analogue chloroacetyl-CoA inhibits fatty acid synthase by reacting with the ‘central’ or pantetheine thiol and not the ‘peripheral’ or β-ketoacylsynthase thiol as previously reported. This was demonstrated by the isolation of [¹⁴C]carboxymethylcysteamine after acid hydrolysis of enzyme labelled with chloro[¹⁴C]acetyl-CoA, and by the demonstration that more than one of the partial reactions is inhibited. This reagent now represents a simple and convenient tool both for quantification of the pantetheine thiol and for labelling this site for peptide mapping and isolation.     

Acetoacetyl-acyl carrier protein synthase. A target for the antibiotic thiolactomycin

The biochemical basis for the inhibition of fatty acid biosynthesis in Escherichia coli by the antibiotic thiolactomycin was investigated. A biochemical assay was developed to measure acetoacetyl-acyl carrier protein (ACP) synthase activity, a recently discovered third condensing enzyme from E. coli (Jackowski, S., and Rock, C.O. (1987) J. Biol. Chem. 262, 7927-7931). In contrast to the other two condensing enzymes in E. coli, acetoacetyl-ACP synthase (synthase III) condensed malonyl-ACP with acetyl-CoA, rather than with acetyl-ACP. The concentration dependence of thiolactomycin inhibition of fatty acid biosynthesis in vivo was the same as the inhibition of acetoacetyl-ACP synthase activity in vitro indicating that the two phenomena were related. A thiolactomycin-resistant mutant (strain CDM5) was isolated. The specific activity of acetoacetyl-ACP synthase in extracts from this mutant was 10-fold lower than in extracts from its thiolactomycin-sensitive parent resulting in a marked defect in the ability of strain CDM5 to incorporate acetyl-CoA into fatty acids in vitro. The residual acetoacetyl-ACP synthase activity in the resistant strain was refractory to thiolactomycin inhibition. In addition, acetyl-CoA:ACP transacylase activity in strain CDM5 was resistant to inactivation by thiolactomycin suggesting that the acetoacetyl-ACP synthase also catalyzes this transacylation reaction. These data point to acetoacetyl-ACP synthase as a target for thiolactomycin inhibition of bacterial fatty acid biosynthesis.     

Purification and characterization of an unusually large fatty acid synthase from Mycobacterium tuberculosis var. bovis BCG.

Fatty acid synthase was purified from Mycobacterium tuberculosis var. bovis BCG. The method developed gave a 23% yield of the synthase and also yielded purified mycocerosic acid synthase. The fatty acid synthase is of unusually large size and composed of two 500-kDa monomers. The amino acid composition of the two synthases was not identical; the N-terminus of the fatty acid synthase was blocked, whereas that of the mycocerosic acid synthase was not. Western blot analysis of crude mycobacterial extracts with polyclonal antibodies prepared against each synthase showed a single band in each case with no cross-reactivity with the other synthase. Fatty acid synthase required both NADH (Km, 11 microM) and NADPH (Km, 14 microM). The Km for acetyl-CoA and malonyl-CoA were 5 and 6 microM, respectively. Fatty acids were released from the synthase as CoA esters. A bimodal distribution of fatty acids was obtained at around C16 and C26. The primer utilization also reflects the de novo synthesis and elongation capabilities of the enzyme; acetyl-CoA was the preferred primer but CoA esters up to C8 but not C12 and C14 could serve as primers, whereas C16 was readily used as a primer for elongation. Addition of CoA and CoA ester-binding oligosaccharides caused enhanced release of C16. Since this mycobacterial fatty acid synthase is twice as large as other multifunctional fatty acid synthases, it is tempting to suggest that this synthase represents a head to tail fusion of two fatty acid synthase genes coding for a double size protein with one-half producing C16 acid and the other elongating the C16 acid to a C26 acid. The monomer of fatty acid synthase from M. smegmatis was immunologically similar and equal in size to the synthase from M. tuberculosis.