Mechanism of the beta-ketoacyl synthase reaction catalyzed by the animal fatty acid synthase

The catalytic mechanism of the beta-ketoacyl synthase domain of the multifunctional fatty acid synthase has been investigated by a combination of mutagenesis, active-site titration, product analysis, and product inhibition. Neither the reactivity of the active-site Cys161 residue toward iodoacetamide nor the rate of unidirectional transfer of acyl moieties to Cys161 was significantly decreased by replacement of any of the conserved residues, His293, His331, or Lys326, with Ala. Decarboxylation of malonyl moieties in the fully-active Cys161Gln background generated equimolar amounts of acetyl-CoA and bicarbonate, rather than carbon dioxide, and was seriously compromised by replacement of any of the conserved basic residues. The ability of bicarbonate to inhibit decarboxylation of malonyl moieties in the Cys161Gln background was significantly reduced by replacement of His293 but less so by replacement of His331. The data are consistent with a reaction mechanism, in which the initial primer transfer reaction is promoted largely through a lowering of the pKa of the Cys161 thiol by a helix dipole effect and activation of the substrate thioester carbon atom by binding of the keto group in an oxyanion hole. The data also indicate that an activated water molecule is present at the active site that is required either for the rapid hydration of carbon dioxide, prior its release as bicarbonate or, alternatively, for an initial attack on the malonyl C3. In the alternative mechanism, a negatively-charged tetrahedral transition state could be generated, stabilized in part by interaction of His293 with the negatively charged oxygen at C3 and interaction of His331 with the negatively charged thioester carbonyl oxygen, that breaks down to generate bicarbonate directly. Finally, the carbanion at C2, attacks the electrophilic C1 of the primer, generating a second tetrahedral transition state, also stabilized through contacts with the oxyanion hole and His331, that breaks down to form the beta-ketoacyl-S-acyl carrier protein product.     

Structural and functional organization of the animal fatty acid synthase

The entire pathway of palmitate synthesis from malonyl-CoA in mammals is catalyzed by a single, homodimeric, multifunctional protein, the fatty acid synthase. Each subunit contains three N-terminal domains, the beta-ketoacyl synthase, malonyl/acetyl transferase and dehydrase separated by a structural core from four C-terminal domains, the enoyl reductase, beta-ketoacyl reductase, acyl carrier protein and thiosterase. The kinetics and specificities of the substrate loading reaction catalyzed by the malonyl/acetyl transferase, the condensation reaction catalyzed by beta-ketoacyl synthase and chain-terminating reaction catalyzed by the thioesterase ensure that intermediates do not leak off the enzyme, saturated chains exclusively are elongated and palmitate is released as the major product. Only in the fatty acid synthase dimer do the subunits adopt conformations that facilitate productive coupling of the individual reactions for fatty acid synthesis at the two acyl carrier protein centers. Introduction of a double tagging and dual affinity chromatographic procedure has permitted the engineering and isolation of heterodimeric fatty acid synthases carrying different mutations on each subunit. Characterization of these heterodimers, by activity assays and chemical cross-linking, has been exploited to map the functional topology of the protein. The results reveal that the two acyl carrier protein domains engage in substrate loading and condensation reactions catalyzed by the malonyl/acetyl transferase and beta-ketoacyl synthase domains of either subunit. In contrast, the reactions involved in processing of the beta-carbon atom, following each chain elongation step, together with the release of palmitate, are catalyzed by the cooperation of the acyl carrier protein with catalytic domains of the same subunit. These findings suggest a revised model for the fatty acid synthase in which the two polypeptides are oriented such that head-to-tail contacts are formed both between and within subunits.     

Mapping the functional topology of the animal fatty acid synthase by mutant complementation in vitro

An in vitro mutant complementation approach has been used to map the functional topology of the animal fatty acid synthase. A series of knockout mutants was engineered, each mutant compromised in one of the seven functional domains, and heterodimers generated by hybridizing all possible combinations of the mutated subunits were isolated and characterized. Heterodimers comprised of a subunit containing either a beta-ketoacyl synthase or malonyl/acetyltransferase mutant, paired with a subunit containing mutations in any one of the other five domains, are active in fatty acid synthesis. Heterodimers in which both subunits carry a knockout mutation in either the dehydrase, enoyl reductase, keto reductase, or acyl carrier protein are inactive. Heterodimers comprised of a subunit containing a thioesterase mutation paired with a subunit containing a mutation in either the dehydrase, enoyl reductase, beta-ketoacyl reductase, or acyl carrier protein domains exhibit very low fatty acid synthetic ability. The results are consistent with a model for the fatty acid synthase in which the substrate loading and condensation reactions are catalyzed by cooperation of an acyl carrier protein domain of one subunit with the malonyl/acetyltransferase or beta-ketoacyl synthase domains, respectively, of either subunit. The beta-carbon-processing reactions, responsible for the complete reduction of the beta-ketoacyl moiety following each condensation step, are catalyzed by cooperation of an acyl carrier protein domain with the beta-ketoacyl reductase, dehydrase, and enoyl reductase domains associated exclusively with the same subunit. The chain-terminating reaction is carried out most efficiently by cooperation of an acyl carrier protein domain with the thioesterase domain of the same subunit. These results are discussed in the context of a revised model for the fatty acid synthase.     

Isolation of a functional transferase component from the rat fatty acid synthase by limited trypsinization of the subunit monomer. Formation of a stable functional complex between transferase and acyl carrier protein domains

Limited trypsinization of rat fatty acid synthase monomers results in cleavage at sites protected in the native dimer. A 47,000-Da polypeptide containing the transferase component was isolated from the digest and its location in the multifunctional polypeptide established. Both acetyl and malonyl moieties are transferred stoichiometrically from CoA ester to this polypeptide and each can replace the other, confirming that a single common site is utilized in the loading of these substrates onto the fatty acid synthase. Transferase activity of the 47,000-Da polypeptide decreases with increasing acyl donor chain length (malonyl = acetyl greater than butyryl greater than hexanoyl greater than octanoyl). Activity is inhibited by certain thiol-directed reagents, and protection is afforded by substrate suggesting the presence of a sensitive cysteine residue near the substrate binding site. The transferase was also able to utilize as acyl acceptor the Escherichia coli acyl carrier protein and the acyl carrier protein domain of the multifunctional fatty acid synthase. When the fatty acid synthase monomer was trypsinized under milder conditions, the 47,000-Da transferase domain could be isolated in association with the 8,000-Da acyl carrier protein domain. The transferase was capable of translocating substrate moieties from CoA ester donors to the associated acyl carrier protein. The results provide the first direct evidence that, in the head-to-tail oriented fatty acid synthase homodimer, functional communication between the transferase domain located near the end of one polypeptide and the acyl carrier protein domain located at the opposite end of the other polypeptide is facilitated by a stable physical interaction between these domains.     

Engineered fatty acid biosynthesis in Streptomyces by altered catalytic function of beta-ketoacyl-acyl carrier protein synthase III

The Streptomyces glaucescens beta-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in S. glaucescens resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D(3) acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII. These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant. To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in Escherichia coli and assayed. In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro. The Cys122Ala mutant exhibited higher activity. This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 microM), a branched-chain fatty acid biosynthetic precursor. Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase. These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within S. glaucescens.     

Fatty acid synthesis. Role of active site histidines and lysine in Cys-His-His-type beta-ketoacyl-acyl carrier protein synthases

Beta-ketoacyl-acyl carrier protein (ACP) synthase enzymes join short carbon units to construct fatty acyl chains by a three-step Claisen condensation reaction. The reaction starts with a trans thioesterification of the acyl primer substrate from ACP to the enzyme. Subsequently, the donor substrate malonyl-ACP is decarboxylated to form a carbanion intermediate, which in the third step attacks C1 of the primer substrate giving rise to an elongated acyl chain. A subgroup of beta-ketoacyl-ACP synthases, including mitochondrial beta-ketoacyl-ACP synthase, bacterial plus plastid beta-ketoacyl-ACP synthases I and II, and a domain of human fatty acid synthase, have a Cys-His-His triad and also a completely conserved Lys in the active site. To examine the role of these residues in catalysis, H298Q, H298E and six K328 mutants of Escherichia colibeta-ketoacyl-ACP synthase I were constructed and their ability to carry out the trans thioesterification, decarboxylation and/or condensation steps of the reaction was ascertained. The crystal structures of wild-type and eight mutant enzymes with and/or without bound substrate were determined. The H298E enzyme shows residual decarboxylase activity in the pH range 6-8, whereas the H298Q enzyme appears to be completely decarboxylation deficient, showing that H298 serves as a catalytic base in the decarboxylation step. Lys328 has a dual role in catalysis: its charge influences acyl transfer to the active site Cys, and the steric restraint imposed on H333 is of critical importance for decarboxylation activity. This restraint makes H333 an obligate hydrogen bond donor at Nepsilon, directed only towards the active site and malonyl-ACP binding area in the fatty acid complex.     

Cloning, expression, and characterization of the human mitochondrial beta-ketoacyl synthase. Complementation of the yeast CEM1 knock-out strain

A human beta-ketoacyl synthase implicated in a mitochondrial pathway for fatty acid synthesis has been identified, cloned, expressed, and characterized. Sequence analysis indicates that the protein is more closely related to freestanding counterparts found in prokaryotes and chloroplasts than it is to the beta-ketoacyl synthase domain of the human cytosolic fatty acid synthase. The full-length nuclear-encoded 459-residue protein includes an N-terminal sequence element of approximately 38 residues that functions as a mitochondrial targeting sequence. The enzyme can elongate acyl-chains containing 2-14 carbon atoms with malonyl moieties attached in thioester linkage to the human mitochondrial acyl carrier protein and is able to restore growth to the respiratory-deficient yeast mutant cem1 that lacks the endogenous mitochondrial beta-ketoacyl synthase and exhibits lowered lipoic acid levels. To date, four components of a putative type II mitochondrial fatty acid synthase pathway have been identified in humans: acyl carrier protein, malonyl transferase, beta-ketoacyl synthase, and enoyl reductase. The substrate specificity and complementation data for the beta-ketoacyl synthase suggest that, as in plants and fungi, in humans this pathway may play an important role in the generation of octanoyl-acyl carrier protein, the lipoic acid precursor, as well as longer chain fatty acids that are required for optimal mitochondrial function.     

Characterization of the beta-carbon processing reactions of the mammalian cytosolic fatty acid synthase: role of the central core

The properties of the beta-ketoacyl reductase, dehydrase, and enoyl reductase components of the animal fatty acid synthase responsible for the reduction of the beta-ketoacyl moiety formed at each round of chain elongation have been studied by engineering and characterizing mutants defective in each of these three catalytic domains. These "beta-carbon processing" mutants leak the stalled four-carbon intermediates by direct transfer to CoA. However, enoyl reductase mutants leak beta-ketobutyryl, beta-hydroxybutyryl, and crotonyl moieties, a finding explained, at least in part, by the observation that the equilibrium and rate constant for the dehydrase reaction favor the formation of beta-hydroxy rather than enoyl moieties. In this regard, the type I animal fatty acid synthase resembles its type II counterpart in Escherichia coli in that both systems rely on the enoyl reductase to pull the beta-carbon processing reactions to completion. Kinetic and nucleotide binding measurements on fatty acid synthases mutated in either of the two nucleotide binding domains revealed that the NADPH binding sites are nonidentical, the enoyl reductase exhibiting higher affinity. Surprisingly, NADPH binding is also completely compromised by certain deletions and mutations in the central core region distant from the nucleotide binding sites. Comparable central core sequences are present in the structurally related modular polyketide synthases, except in those modules that lack all three beta-carbon processing enzymes. These findings suggest that the central core region of fatty acid and polyketide synthases plays an important role in facilitating the beta-carbon processing reactions.     

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase

Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.     

Methylmalonyl coenzyme A selectivity of cloned and expressed acyltransferase and beta-ketoacyl synthase domains of mycocerosic acid synthase from Mycobacterium bovis BCG.

Methyl-branched fatty acids and polyketides occur in a variety of living organisms. Previous studies have established that multifunctional enzymes use methylmalonyl coenzyme A (CoA) as the substrate to generate methyl-branched products such as mycocerosic acids and polyketides. However, we do not know which of the component activities show selectivity for methylmalonyl-CoA in any biological system. A comparison of homologies of the domains of the multifunctional synthases that selectively use malonyl-CoA or methylmalonyl-CoA suggested that the acyltransferase (AT) and beta-ketoacyl synthase (KS) domains might be responsible for the substrate selectivity. To test this hypothesis, we expressed the AT and KS domains of the mycocerosic acid synthase (MAS) gene from Mycobacterium bovis BCG in Escherichia coli and examined whether they confer to synthases that normally do not use methylmalonyl-CoA the ability to incorporate methylmalonyl-CoA into fatty acids. Both the AT and the KS domains of MAS showed selectivity for methylmalonyl-CoA over malonyl-CoA. Acyl carrier protein (ACP)-dependent elongation of the n-C12 acyl primer mainly by one methylmalonyl-CoA unit was catalyzed by an E. coli fatty acid synthase preparation only in the presence of the expressed MAS domains. An ACP-dependent elongation of the n-C20 acyl primer by one methylmalonyl-CoA extender unit was catalyzed by fatty acid synthase from Mycobacterium smegmatis only in the presence of the expressed MAS domains. These results show methylmalonyl-CoA selectivity for the AT and KS domains of MAS. These domains may be useful in producing novel polyketides by genetic engineering.     

鸭肝脂肪酸合成酶的NADPH底物抑制及作用动力学

己知动物脂肪酸合成酶的底物乙酰辅酶Ａ和丙二酰辅酶Ａ具有竞争性双底物抑制的乒乓机制。实验发现鸭肝脂肪酸合成酶的第三个底物ＮＡＤＰＨ也具有底物抑制,并研究了它的规律及与ＮＡＤＰＨ有关的稳态动力学。发现对于该酶的全反应,增加丙二酰辅酶Ａ浓度,降低环境盐浓度,均使ＮＡＤＰＨ底物抑制减少。但以ＮＡＤＰＨ作底物的酮酰还原和烯酰还原二步单独反应以及包含四步单独反应的乙酰乙酰辅酶Ａ还原反应都无ＮＡＤＰＨ底物抑制现象。ＮＡＤＰＨ底物抑制对丙二酰辅酶Ａ为竞争性,丙二酰辅酶Ａ底物抑制对ＮＡＤＰＨ为非竞争性。在全反应中ＮＡＤＰＨ和丙二酰辅酶Ａ之间发现为乒乓机制,在乙酰乙酰辅酶Ａ还原反应中,两个底物ＮＡＤＰＨ和乙酰乙酰辅酶Ａ之间则表现为序列反应机制。降低环境盐浓度使ＮＡＤＰＨ和丙二酰辅酶Ａ之间的乒乓机制向序列机制转化。在全反应中,ＮＡＤＰ产物抑制相对ＮＡＤＰ为竞争性,对丙二酰辅酶Ａ为非竞争性。     

Crystal structure of beta-ketoacyl-acyl carrier protein synthase II from E.coli reveals the molecular architecture of condensing enzymes.

In the biosynthesis of fatty acids, the beta-ketoacyl-acyl carrier protein (ACP) synthases catalyze chain elongation by the addition of two-carbon units derived from malonyl-ACP to an acyl group bound to either ACP or CoA. The crystal structure of beta-ketoacyl synthase II from Escherichia coli has been determined with the multiple isomorphous replacement method and refined at 2.4 A resolution. The subunit consists of two mixed five-stranded beta-sheets surrounded by alpha-helices. The two sheets are packed against each other in such a way that the fold can be described as consisting of five layers, alpha-beta-alpha-beta-alpha. The enzyme is a homodimer, and the subunits are related by a crystallographic 2-fold axis. The two active sites are located near the dimer interface but are approximately 25 A apart. The proposed nucleophile in the reaction, Cys163, is located at the bottom of a mainly hydrophobic pocket which is also lined with several conserved polar residues. In spite of very low overall sequence homology, the structure of beta-ketoacyl synthase is similar to that of thiolase, an enzyme involved in the beta-oxidation pathway, indicating that both enzymes might have a common ancestor.     

Dissecting the component reactions catalyzed by the actinorhodin minimal polyketide synthase

The actinorhodin (act) minimal polyketide synthase (PKS) from Streptomyces coelicolor consists of three proteins: an acyl carrier protein (ACP) and two beta-ketoacyl ACP synthase components known as KSalpha and KSbeta. The act minimal PKS catalyzes at least 18 separate reactions which can be divided into loading, initiation, extension, and cyclization and release phases. Two quantitative kinetic assays were developed and used to measure individual rate and Michaelis constants for loading, initiation and extension steps. In the minimal PKS, the reaction between malonyl CoA and ACP to form malonyl ACP (loading) is the rate-limiting step (kcat = 0.49 min-1, KM = 207 microM). This reaction increases 5-fold in rate in the presence of KSalphaKSbeta (kcat = 2.3 min-1, KM = 215 microM). In the presence of S. coelicolor malonyl CoA:ACP transacylase (MCAT), the rate of loading increases and the kinetic parameters of malonyl-ACP as a substrate of KSalphaKSbeta can be measured (kcat = 20.6 min-1, KM = 2.4 microM). Under these conditions, it appears that decarboxylation of malonyl-ACP to form acetyl-ACP (initiation) is the rate-limiting step. When an excess of acetyl ACP is supplied, either chain extension, cyclization, or release steps become rate limiting (k approximately 60 min-1). No ACP-bound intermediates could be observed, suggesting that partially or fully extended chains do not accumulate because chain extension is rate limiting under these conditions and that cyclization and release are fast. apo-ACP acts as a mixed inhibitor of malonyl ACP binding to KSalpha/KSbeta (Kic = 50 microM, Kiu = 137 microM), but apo-ACP does not appear to inhibit MCAT.     

The yeast fatty acid synthase. Pathway for transfer of the acetyl group from coenzyme A to the Cys-SH of the condensation site

The reaction pathway of enzyme-catalyzed acetylation of the acyl-accepting sites of the yeast synthase, a Ser-OH at the acetyl transacylase site, a Cys-SH at the beta-ketoacyl synthase site, and the acyl carrier protein 4'-phosphopantetheine-SH (Pant-SH), has been investigated using the chromophoric substrate, p-nitrophenyl thioacetate. The stoichiometry of acetylation of the native enzyme was 3 mol of acetate bound per mol of synthase unit, alpha beta (Mr 430,000). The acetylation process is biexponential; the rate constant of acetylation of the first 2 mol is 5.0 s-1 and the third mol is 0.2 s-1. The pathway by which acetyl moiety is added to the enzyme was determined by selectively blocking the acyl-accepting sites and subsequently determining the kinetics and stoichiometry of acetylation. The dibromopropanone-treated enzyme, in which the Pant-SH and Cys-SH are alkylated, exhibited an exponential burst of approximately 1 mol/mol of synthase unit with a rate constant of 11.0 s-1. The iodoacetamide-treated enzyme, in which Cys-SH is alkylated, had a biexponential burst with a total stoichiometry of approximately 2 mol/mol of synthase unit, with rate constants of 9 and 0.2 s-1, respectively. The kinetically competent acetylation to the extent of 2 and approximately 1 mol/mol of synthase unit for both Cys-SH and Cys-SH and Pant-SH-blocked enzymes, respectively, indicated that the route of acetyl transfer in the yeast synthase is obligatorily Ser-OH----Cys-SH. The acetylation of Pant-SH (0.2 s-1) occurs with a rate insignificant to the process of fatty acid synthesis (turnover rate constant of 1.5 s-1). These conclusions are supported by experiments involving end point radiolabeling of the synthase with [1-14C]acetyl moieties using the substrate, p-nitrophenyl thio[1-14C]acetate. Native, dibromopropanone-treated, and iodoacetamide-treated enzymes bind about 3, 1, and 2 mol of acetyl/mol of synthase unit, respectively. Performic acid oxidation studies of the acetyl-labeled enzyme indicate that there is one Ser-O-acetyl formed in the native and alkylated enzymes and one Cys-S-acetyl and one Pant-S-acetyl formed in the native enzyme. Altogether, these results support our contention that the acetylation of the Pant-SH is kinetically incompetent. Thus, the yeast synthase transacetylation reactions occur by a novel process of acetyl transfer from CoA to Ser-OH----Cys-SH, which is in contrast to the transfer from CoA to Ser-OH----Pant-SH----Cys-SH catalyzed by the prokaryotic synthases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris.

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.     

Dibromopropanone cross-linking of the phosphopantetheine and active-site cysteine thiols of the animal fatty acid synthase can occur both inter- and intrasubunit. Reevaluation of the side-by-side, antiparallel subunit model

The objective of this study was to test a new model for the homodimeric animal FAS which implies that the condensation reaction can be catalyzed by the amino-terminal beta-ketoacyl synthase domain in cooperation with the penultimate carboxyl-terminal acyl carrier protein domain of either subunit. Treatment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; none of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the beta-ketoacyl synthase domain (C161A) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151A). A double affinity-labeling strategy was used to isolate dimers that carried one or both mutations on one or both subunits; the heterodimers were treated with dibromopropanone and analyzed by a combination of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtration, and matrix-assisted laser desorption mass spectrometry. Thus the two slowest moving of these species, which accounted for 45 and 15% of the total, were identified as doubly and singly cross-linked dimers, respectively, whereas the fastest moving species, which accounted for 35% of the total, was identified as originating from internally cross-linked subunits. These results show that the two polypeptides of the fatty acid synthase are oriented such that head-to-tail contacts are formed both between and within subunits, and provide the first structural evidence in support of the new model.     

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.     

Enzymatic formation of a resorcylic acid by creating a structure‐guided single‐point mutation in stilbene synthase

A novel C17 resorcylic acid was synthesized by a structure‐guided Vitis vinifera stilbene synthase (STS) mutant, in which threonine 197 was replaced with glycine (T197G). Altering the architecture of the coumaroyl binding and cyclization pocket of the enzyme led to the attachment of an extra acetyl unit, derived from malonyl‐CoA, to p‐coumaroyl‐CoA. The resulting novel pentaketide can be produced strictly by STS‐like enzymes and not by Chalcone synthase‐like type III polyketide synthases; due to the unique thioesterase like activity of STS‐like enzymes. We utilized a liquid chromatography mass spectrometry‐based data analysis approach to directly compare the reaction products of the mutant and wild type STS. The findings suggest an easy to employ platform for precursor‐directed biosynthesis and identification of unnatural polyketides by structure‐guided mutation of STS‐like enzymes.     

Kinetic and mechanistic analysis of the malonyl CoA:ACP transacylase from Streptomyces coelicolor indicates a single catalytically competent serine nucleophile at the active site

The source of malonyl groups for polyketide and fatty acid biosynthesis is malonyl CoA. During fatty acid and polyketide biosynthesis, malonyl groups are normally transferred to the acyl carrier protein (ACP) component of the synthase by a malonyl CoA:holo-ACP transacylase (MCAT) enzyme. The fatty acid synthase (FAS) malonyl CoA:ACP transacylase from Streptomyces coelicolor was expressed in Escherichia coli as a hexahistidine-tagged (His(6)) fusion protein in high yield. The His(6)-MCAT was purified to homogeneity using standard techniques, and kinetic analysis of the malonylation of S. coelicolorFAS holo-ACP, catalyzed by His(6)-MCAT, gave K(infinity) (M) values of 73 (ACP) and 60 microM (malonyl CoA). A catalytic constant k (infinity) (M) of 450 s(-1) and specificity constants k (infinity) (M)/K (infinity) (M) of 6.2 (ACP) and 7.5 microM(-1) s(-1) (malonyl CoA) were measured. Malonyl transfer to the E. coli FAS holo-ACP, catalyzed by His(6)-MCAT, was less efficient (k (infinity) (M)/K (infinity) (M) was 10% of that of the S. coelicolor ACP). Incubation of MCAT with the serine specific agent PMSF caused inhibition of malonyl transfer to FAS ACPs, and an S97A MCAT mutant was incapable of catalyzing malonyl transfer. Our results show that in the reaction with FAS holo-ACPs the S. coelicolor MCAT is very similar to the E. coli MCAT paradigm in terms of its kinetic mechanism and active site residues. These results indicate that no other active site nucleophile is involved in catalysis as has been suggested to explain recently reported observations.     

Cloning,expression, characterization,and interaction of two components of a human mitochondrial fatty acid synthase. Malonyltransferase and acyl carrier protein

The possibility that human cells contain, in addition to the cytosolic type I fatty acid synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of fatty acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonyl-CoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/acetyltransferase component of the cytosolic type I fatty acid synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I fatty acid synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonyl-CoA/acyl carrier protein-dependent fatty acid synthase system, distinct from the type I cytosolic fatty acid synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function.