Effect of modification of the length and flexibility of the acyl carrier protein-thioesterase interdomain linker on functionality of the animal fatty acid synthase

A natural linker of approximately 20 residues connects the acyl carrier protein with the carboxy-terminal thioesterase domain of the animal fatty acid synthase. This study examines the effects of changes in the length and amino acid composition of this linker on catalytic activity, product composition, and segmental motion of the thioesterase domain. Deletion of 10 residues, almost half of the interdomain linker, had no effect on either mobility of the thioesterase domain, estimated from fluorescence polarization of a pyrenebutyl methylphosphono moiety bound covalently to the active site serine residue, or functionality of the fatty acid synthase; further shortening of the linker limited mobility of the thioesterase domain and resulted in reduced fatty acid synthase activity and an increase in product chain length from 16 to 18 and 20 carbon atoms. Surprisingly, however, even when the entire linker region was deleted, the fatty acid synthase retained 28% activity. Lengthening of the linker, by insertion of an unusually long acyl carrier protein-thioesterase linker from a modular polyketide synthase, increased mobility of the thioesterase domain without having any significant effect on catalytic properties of the complex. Interdomain linkers could also be used to tether, to the acyl carrier protein domain of the fatty acid synthase, a thioesterase active toward shorter chain length acyl thioesters generating novel short-chain fatty acid synthases. These studies reveal that although truncation of the interdomain linker partially impacts the ability of the thioesterase domain to terminate growth of the acyl chain, the overall integrity of the fatty acid synthase is quite tolerant to moderate changes in linker length and flexibility. The retention of fatty acid synthesizing activity on deletion of the entire linker region implies that the inherent flexibility of the phosphopantetheine "swinging arm" also contributes significantly to the successful docking of the long-chain acyl moiety in the thioesterase active site.     

Terminal alkene formation by the thioesterase of curacin A biosynthesis: structure of a decarboxylating thioesterase

Curacin A is a polyketide synthase (PKS)-non-ribosomal peptide synthetase-derived natural product with potent anticancer properties generated by the marine cyanobacterium Lyngbya majuscula. Type I modular PKS assembly lines typically employ a thioesterase (TE) domain to off-load carboxylic acid or macrolactone products from an adjacent acyl carrier protein (ACP) domain. In a striking departure from this scheme the curacin A PKS employs tandem sulfotransferase and TE domains to form a terminal alkene moiety. Sulfotransferase sulfonation of β-hydroxy-acyl-ACP is followed by TE hydrolysis, decarboxylation, and sulfate elimination (Gu, L., Wang, B., Kulkarni, A., Gehret, J. J., Lloyd, K. R., Gerwick, L., Gerwick, W. H., Wipf, P., Håkansson, K., Smith, J. L., and Sherman, D. H. (2009) J. Am. Chem. Soc. 131, 16033–16035). With low sequence identity to other PKS TEs (<15%), the curacin TE represents a new thioesterase subfamily. The 1.7-Å curacin TE crystal structure reveals how the familiar α/β-hydrolase architecture is adapted to specificity for β-sulfated substrates. A Ser-His-Glu catalytic triad is centered in an open active site cleft between the core domain and a lid subdomain. Unlike TEs from other PKSs, the lid is fixed in an open conformation on one side by dimer contacts of a protruding helix and on the other side by an arginine anchor from the lid into the core. Adjacent to the catalytic triad, another arginine residue is positioned to recognize the substrate β-sulfate group. The essential features of the curacin TE are conserved in sequences of five other putative bacterial ACP-ST-TE tridomains. Formation of a sulfate leaving group as a biosynthetic strategy to facilitate acyl chain decarboxylation is of potential value as a route to hydrocarbon biofuels.     

Enzymatic assembly of epothilones: the EpoC subunit and reconstitution of the EpoA-ACP/B/C polyketide and nonribosomal peptide interfaces

The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value. Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli. EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis. The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction. The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB. The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain. The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible. These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.     

Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine.

beta-Ketoacyl synthases involved in the biosynthesis of fatty acids and polyketides exhibit extensive sequence similarity and share a common reaction mechanism, in which the carbanion participating in the condensation reaction is generated by decarboxylation of a malonyl or methylmalonyl moiety; normally, the decarboxylation step does not take place readily unless an acyl moiety is positioned on the active-site cysteine residue in readiness for the ensuing condensation reaction. Replacement of the cysteine nucleophile (Cys-161) with glutamine, in the beta-ketoacyl synthase domain of the multifunctional animal fatty acid synthase, completely inhibits the condensation reaction but increases the uncoupled rate of malonyl decarboxylation by more than 2 orders of magnitude. On the other hand, replacement with Ser, Ala, Asn, Gly, and Thr compromises the condensation reaction without having any marked effect on the decarboxylation reaction. The affinity of the beta-ketoacyl synthase for malonyl moieties, in the absence of acetyl moieties, is significantly increased in the Cys161Gln mutant compared to that in the wild type and is similar to that exhibited by the wild-type beta-ketoacyl synthase in the presence of an acetyl primer. These results, together with modeling studies of the Cys --> Gln mutant from the crystal structure of the Escherichia coli beta-ketoacyl synthase II enzyme, suggest that the side chain carbonyl group of the Gln-161 can mimic the carbonyl of the acyl moiety in the acyl-enzyme intermediate so that the mutant adopts a conformation analogous to that of the acyl-enzyme intermediate. Catalysis of the decarboxylation of malonyl-CoA requires the dimeric form of the Cys161Gln fatty acid synthase and involves prior transfer of the malonyl moiety from the CoA ester to the acyl carrier protein domain and subsequent release of the acetyl product by transfer back to a CoA acceptor. These results suggest that the role of the Cys --> Gln beta-ketoacyl synthases found in the loading domains of some modular polyketide synthases likely is to act as malonyl, or methylmalonyl, decarboxylases that provide a source of primer for the chain extension reactions catalyzed by associated modules containing fully competent beta-ketoacyl synthases.     

A new substrate specificity for acyl transferase domains of the ascomycin polyketide synthase in Streptomyces hygroscopicus.

Ascomycin (FK520) is a structurally complex macrolide with immunosuppressant activity produced by Streptomyces hygroscopicus. The biosynthetic origin of C12-C15 and the two methoxy groups at C13 and C15 has been unclear. It was previously shown that acetate is not incorporated into C12-C15 of the macrolactone ring. Here, the acyl transferase (AT) of domain 8 in the ascomycin polyketide synthase was replaced with heterologous ATs by double homologous recombination. When AT8 was replaced with methylmalonyl-CoA-specific AT domains, the strains produced 13-methyl-13-desmethoxyascomycin, whereas when AT8 was replaced with a malonyl-specific domain, the strains produced 13-desmethoxyascomycin. These data show that ascomycin AT8 does not use malonyl- or methylmalonyl-CoA as a substrate in its native context. Therefore, AT8 must be specific for a substrate bearing oxygen on the alpha carbon. Feeding experiments showed that [(13)C]glycerol is incorporated into C12-C15 of ascomycin, indicating that both modules 7 and 8 of the polyketide synthase use an extender unit that can be derived from glycerol. When AT6 of the 6-deoxyerythronolide B synthase gene was replaced with ascomycin AT8 and the engineered gene was expressed in Streptomyces lividans, the strain produced 6-deoxyerythronolide B and 2-demethyl-6-deoxyerythronolide B. Therefore, although neither malonyl-CoA nor methylmalonyl-CoA is a substrate for ascomycin AT8 in its native context, both are substrates in the foreign context of the 6-deoxyerythronolide B synthase. Thus, we have demonstrated a new specificity for an AT domain in the ascomycin polyketide synthase and present evidence that specificity can be affected by context.     

Slow Onset Inhibition of Bacterial ��-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin

Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the β-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices α5 and α6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100–200 μg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton.     

Quantitative analysis of loading and extender acyltransferases of modular polyketide synthases

The acyltransferase (AT) domains of modular polyketide synthases (PKSs) are the primary determinants of building block specificity in polyketide biosynthesis and are therefore attractive targets for protein engineering. Thus far, investigations into the fundamental biochemical properties of AT domains have been hampered by the inability to produce these enzymes as self-standing polypeptides. Here we describe an alternative, generally applicable strategy for overexpression and analysis of AT domains from modular PKSs as truncated didomain proteins (approximately 60 kDa). Recently, we reported the expression and reconstitution of the loading didomain of 6-deoxyerythronolide B synthase (Lau, J., Cane, D. E., and Khosla, C. (2000) Biochemistry 39, 10514-20). By replacing the AT domain of this protein with a methylmalonyl-CoA specific AT domain from module 6 of the 6-deoxyerythronolide B synthase, or alternatively a malonyl-CoA specific AT domain from module 2 of the rapamycin synthase, each of these extender unit AT domains could be overproduced and purified to homogeneity. Using acyl-CoA substrates as acyl group donors and N-acetylcysteamine as the thiol acceptor, we devised a steady-state kinetic assay to probe the properties of these three didomain proteins and selected mutants. Propionyl-CoA was the preferred substrate of the loading didomain, although acetyl- and butyryl-CoA were also accepted with approximately 40-fold-lower specificity. In contrast to the relatively relaxed specificity of the loading AT domain, the methylmalonyl- and malonyl-specific AT domains had high specificity (>1000-fold) toward their natural substrates. The acyl transfer reaction was inhibited by coenzyme A (CoASH) with both a competitive and a noncompetitive component. Use of an exogenous holo-acyl carrier protein (ACP) as an acceptor thiol did not increase the rate of acyl transfer relative to the reaction involving N-acetylcysteamine, suggesting that either the on-rate of the acyl group is rate-limiting or that the apo-ACP component of the didomain protein precludes effective docking of a second ACP onto the AT active site. Mutation of Trp-222 in the loading AT domain to an Arg residue that is universally conserved in all extender unit AT domains failed to enable the loading AT domain to accept methylmalonyl-CoA as an alternative substrate. In contrast, mutation of the equivalent Arg residue in an extender AT domain resulted in a protein with no activity. Together, these results provide a foundation for future structural and mechanistic investigations into the properties of AT domains of modular PKSs.     

Structural and biochemical elucidation of mechanism for decarboxylative condensation of beta-keto acid by curcumin synthase

The typical reaction catalyzed by type III polyketide synthases (PKSs) is a decarboxylative condensation between acyl-CoA (starter substrate) and malonyl-CoA (extender substrate). In contrast, curcumin synthase 1 (CURS1), which catalyzes curcumin synthesis by condensing feruloyl-CoA with a diketide-CoA, uses a β-keto acid (which is derived from diketide-CoA) as an extender substrate. Here, we determined the crystal structure of CURS1 at 2.32 Å resolution. The overall structure of CURS1 was very similar to the reported structures of type III PKSs and exhibited the αβαβα fold. However, CURS1 had a unique hydrophobic cavity in the CoA-binding tunnel. Replacement of Gly-211 with Phe greatly reduced the enzyme activity. The crystal structure of the G211F mutant (at 2.5 Å resolution) revealed that the side chain of Phe-211 occupied the hydrophobic cavity. Biochemical studies demonstrated that CURS1 catalyzes the decarboxylative condensation of a β-keto acid using a mechanism identical to that for normal decarboxylative condensation of malonyl-CoA by typical type III PKSs. Furthermore, the extender substrate specificity of CURS1 suggested that hydrophobic interaction between CURS1 and a β-keto acid may be important for CURS1 to use an extender substrate lacking the CoA moiety. From these results and a modeling study on substrate binding, we concluded that the hydrophobic cavity is responsible for the hydrophobic interaction between CURS1 and a β-keto acid, and this hydrophobic interaction enables the β-keto acid moiety to access the catalytic center of CURS1 efficiently.     

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃‐S‐ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.     

Rigidifying Acyl Carrier Protein Domain in Iterative Type I PKS CalE8 Does Not Affect Its Function

Acyl carrier protein (ACP) domains shuttle acyl intermediates among the catalytic domains of multidomain type I fatty acid synthase and polyketide synthase (PKS) systems. It is believed that the unique function of ACPs is associated with their dynamic property, but it remains to be fully elucidated what type of protein dynamics is critical for the shuttling domain. Using NMR techniques, we found that the ACP domain of iterative type I PKS CalE8 from Micromonospora echinospora is highly dynamic on the millisecond-second timescale. Introduction of an interhelical disulfide linkage in the ACP domain suppresses the dynamics on the millisecond-second timescale and reduces the mobility on the picosecond-nanosecond timescale. We demonstrate that the full-length PKS is fully functional upon rigidification of the ACP domain, suggesting that although the flexibility of the disordered terminal linkers may be important for the function of the ACP domain, the internal dynamics of the helical regions is not critical for that function.     

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.     

Induced-fit upon ligand binding revealed by crystal structures of the hot-dog fold thioesterase in dynemicin biosynthesis

Dynemicins are structurally related 10-membered enediyne natural products isolated from Micromonospora chernisa with potent antitumor and antibiotic activity. The early biosynthetic steps of the enediyne moiety of dynemicins are catalyzed by an iterative polyketide synthase (DynE8) and a thioesterase (DynE7). Recent studies indicate that the function of DynE7 is to off-load the linear biosynthetic intermediate assembled on DynE8. Here, we report crystal structures of DynE7 in its free form at 2.7 Å resolution and of DynE7 in complex with the DynE8-produced all-trans pentadecen-2-one at 2.1 Å resolution. These crystal structures reveal that upon ligand binding, significant conformational changes throughout the substrate-binding tunnel result in an expanded tunnel that traverses an entire monomer of the tetrameric DynE7 protein. The enlarged inner segment of the channel binds the carbonyl-conjugated polyene mainly through hydrophobic interactions, whereas the putative catalytic residues are located in the outer segment of the channel. The crystallographic information reinforces an unusual catalytic mechanism that involves a strictly conserved arginine residue for this subfamily of hot-dog fold thioesterases, distinct from the typical mechanism for hot-dog fold thioesterases that utilizes an acidic residue for catalysis.     

Role of a conserved arginine residue in linkers between the ketosynthase and acyltransferase domains of multimodular polyketide synthases

The role of interdomain linkers in modular polyketide synthases is poorly understood. Analysis of the 6-deoxyerythronolide B synthase (DEBS) has yielded a model in which chain elongation is governed by interactions between the acyl carrier protein domain and the ketosynthase domain plus an adjacent linker. Alanine scanning mutagenesis of the conserved residues of this linker in DEBS module 3 led to the identification of the R513A mutant with a markedly reduced rate of chain elongation. Limited proteolysis supported a structural role for this Arg. Our findings highlight the importance of domain-linker interactions in assembly line polyketide biosynthesis.     

Aspergillus oryzae CsyB Catalyzes the Condensation of Two β-Ketoacyl-CoAs to Form 3-Acetyl-4-hydroxy-6-alkyl-α-pyrone

The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C₉–C₁₇ in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs.     

A Polyketide Synthase Acyltransferase Domain Structure Suggests a Recognition Mechanism for Its Hydroxymalonyl-Acyl Carrier Protein Substrate

We have previously shown that the acyl transferase domain of ZmaA (ZmaA-AT) is involved in the biosynthesis of the aminopolyol polyketide/nonribosomal peptide hybrid molecule zwittermicin A from cereus UW85, and that it specifically recognizes the precursor hydroxymalonyl-acyl carrier protein (ACP) and transfers the hydroxymalonyl extender unit to a downstream second ACP via a transacylated AT domain intermediate. We now present the X-ray crystal structure of ZmaA-AT at a resolution of 1.7 Å. The structure shows a patch of solvent-exposed hydrophobic residues in the area where the AT is proposed to interact with the precursor ACP. We addressed the significance of the AT/ACP interaction in precursor specificity of the AT by testing whether malonyl- or methylmalonyl-ACP can be recognized by ZmaA-AT. We found that the ACP itself biases extender unit selection. Until now, structural information for ATs has been limited to ATs specific for the CoA-linked precursors malonyl-CoA and (2S)-methylmalonyl-CoA. This work contributes to polyketide synthase engineering efforts by expanding our knowledge of AT/substrate interactions with the structure of an AT domain that recognizes an ACP-linked substrate, the rare hydroxymalonate. Our structure suggests a model in which ACP interaction with a hydrophobic motif promotes secondary structure formation at the binding site, and opening of the adjacent substrate pocket lid to allow extender unit binding in the AT active site.     

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.     

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.     

FT-ICR-MS characterization of intermediates in the biosynthesis of the α-methylbutyrate side chain of lovastatin by the 277 kDa polyketide synthase LovF

There are very few fungal polyketide synthases that have been characterized by mass spectrometry. In this paper we describe the in vitro reconstitution and FT-ICR-MS verification of the full activity of an intact 277 kDa fungal polyketide synthase LovF of the lovastatin biosynthetic pathway. We report here both the verification of the reconstitution of fully functional holo-LovF by using (13)C-labeled malonyl-CoA to form α-methylbutyrate functionality and also detection of five predicted intermediates covalently bound to the 4'-phosphopantetheine at the acyl carrier protein (ACP) active site utilizing the phosphopantetheine ejection assay and high-resolution mass spectrometry. Under in vitro conditions, the diketide acetoacetyl intermediate did not accumulate on the ACP active site of holo-LovF following incubation with malonyl-CoA substrate. We found that incubation of holo-LovF with acetoacetyl-CoA served as an effective means of loading the diketide intermediate onto the ACP active site of LovF. Our results demonstrate that subsequent α-methylation of the acetoacetyl intermediate stabilizes the intermediate onto the ACP active site and facilitates the formation and mass spectrometric detection of additional intermediates en route to the formation of α-methylbutyrate.     

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.