Novel intermolecular iterative mechanism for biosynthesis of mycoketide catalyzed by a bimodular polyketide synthase

In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-beta-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel "modularly iterative" mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.     

Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates

In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate. THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused ring tetrahydroxynaphthalene (THN) skeleton. The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood. However, the mechanistic basis for the unusual production of THN and dual cyclization of its malonyl-primed pentaketide is obscure. Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in facilitating malonyl-primed polyketide extension beyond the triketide stage. The resulting new mechanistic model, involving the use of additional cysteines to alter and steer polyketide reactivity, may generally apply to other PKS reaction mechanisms, including those catalyzed by iterative type I and II PKS enzymes. Our crystal structure also reveals an unanticipated novel cavity extending into the "floor" of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter. This tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation.     

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.     

Kinetic analysis of the actinorhodin aromatic polyketide synthase.

Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [(14)C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.     

The acyltransferase homologue from the initiation module of the R1128 polyketide synthase is an acyl-ACP thioesterase that edits acetyl primer units

Type II polyketide synthases (PKSs) synthesize polyfunctional aromatic polyketides through iterative condensations of malonyl extender units. The biosynthesis of most aromatic polyketides is initiated through an acetate unit derived from decarboxylation of malonyl-acyl carrier protein (ACP). Modification of this primer unit represents a powerful method of generating novel polyketides. We have demonstrated that recombination of the initiation module from the R1128 PKS with heterologous elongation modules afforded regioselectively modified polyketides containing alternative primer units. With the exception of the role of the acyltransferase homologue ZhuC, the catalytic cycle of the initiation module has been well explored. ZhuC, along with the ketosynthase III homologue ZhuH and the ACP(p) ZhuG, is essential for the in vivo biosynthesis of aromatic polyketides derived from non-acetate primer units. Here we have studied the role of ZhuC using PKS proteins reconstituted in vitro. We show that the tetracenomycin (tcm) minimal PKS can be directly primed with non-acetate acyl groups. In the presence of approximately 10 microM hexanoyl-ZhuG or approximately 100 microM hexanoyl-CoA, the tcm minimal PKS synthesized hexanoyl-primed analogues of octaketides SEK4 and SEK4b, as well as acetate-primed decaketides SEK15 and SEK15b at comparable levels. Addition of ZhuC abolished synthesis of the acetate-primed decaketides, resulting in exclusive synthesis of the hexanoyl-primed octaketides. In the absence of alternative acyl donors, ZhuC severely retarded the activity of the tcm minimal PKS. The editing capabilities of ZhuC were directly revealed by demonstrating that ZhuC has 100 times greater specificity for acetyl- and propionyl-ACP as compared to hexanoyl- and octanoyl-ACP. Thus, by purging the acetate primer units that otherwise dominate polyketide chain initiation, ZhuC (and presumably its homologues in other PKSs such as the doxorubicin and frenolicin PKSs) allows alternative primer units to be utilized by the elongation module in vivo. The abilities of other alkylacyl primer units to prime the tcm minimal PKS were also investigated in this report.     

Structures of beta-ketoacyl-acyl carrier protein synthase I complexed with fatty acids elucidate its catalytic machinery

BACKGROUND: beta-ketoacyl-acyl carrier protein synthase (KAS) I is vital for the construction of the unsaturated fatty acid carbon skeletons characterizing E. coli membrane lipids. The new carbon-carbon bonds are created by KAS I in a Claisen condensation performed in a three-step enzymatic reaction. KAS I belongs to the thiolase fold enzymes, of which structures are known for five other enzymes. RESULTS: Structures of the catalytic Cys-Ser KAS I mutant with covalently bound C10 and C12 acyl substrates have been determined to 2.40 and 1.85 A resolution, respectively. The KAS I dimer is not changed by the formation of the complexes but reveals an asymmetric binding of the two substrates bound to the dimer. A detailed model is proposed for the catalysis of KAS I. Of the two histidines required for decarboxylation, one donates a hydrogen bond to the malonyl thioester oxo group, and the other abstracts a proton from the leaving group. CONCLUSIONS: The same mechanism is proposed for KAS II, which also has a Cys-His-His active site triad. Comparison to the active site architectures of other thiolase fold enzymes carrying out a decarboxylation step suggests that chalcone synthase and KAS III with Cys-His-Asn triads use another mechanism in which both the histidine and the asparagine interact with the thioester oxo group. The acyl binding pockets of KAS I and KAS II are so similar that they alone cannot provide the basis for their differences in substrate specificity.     

High resolution reaction intermediates of rabbit muscle fructose-1,6-bisphosphate aldolase: substrate cleavage and induced fit

Crystal structures were determined to 1.8 A resolution of the glycolytic enzyme fructose-1,6-bis(phosphate) aldolase trapped in complex with its substrate and a competitive inhibitor, mannitol-1,6-bis(phosphate). The enzyme substrate complex corresponded to the postulated Schiff base intermediate and has reaction geometry consistent with incipient C3-C4 bond cleavage catalyzed Glu-187, which is adjacent by to the Schiff base forming Lys-229. Atom arrangement about the cleaved bond in the reaction intermediate mimics a pericyclic transition state occurring in nonenzymatic aldol condensations. Lys-146 hydrogen-bonds the substrate C4 hydroxyl and assists substrate cleavage by stabilizing the developing negative charge on the C4 hydroxyl during proton abstraction. Mannitol-1,6-bis(phosphate) forms a noncovalent complex in the active site whose binding geometry mimics the covalent carbinolamine precursor. Glu-187 hydrogen-bonds the C2 hydroxyl of the inhibitor in the enzyme complex, substantiating a proton transfer role by Glu-187 in catalyzing the conversion of the carbinolamine intermediate to Schiff base. Modeling of the acyclic substrate configuration into the active site shows Glu-187, in acid form, hydrogen-bonding both substrate C2 carbonyl and C4 hydroxyl, thereby aligning the substrate ketose for nucleophilic attack by Lys-229. The multifunctional role of Glu-187 epitomizes a canonical mechanistic feature conserved in Schiff base-forming aldolases catalyzing carbohydrate metabolism. Trapping of tagatose-1,6-bis(phosphate), a diastereoisomer of fructose 1,6-bis(phosphate), displayed stereospecific discrimination and reduced ketohexose binding specificity. Each ligand induces homologous conformational changes in two adjacent alpha-helical regions that promote phosphate binding in the active site.     

The first 28 N-terminal amino acid residues of human heart muscle carnitine palmitoyltransferase I are essential for malonyl CoA sensitivity and high-affinity binding

Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30-100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues. All of the deletions except Delta83 were active. Mitochondria from the yeast strains expressing Delta28 and Delta39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding. The high-affinity site (K(D1), B(max1)) for binding of malonyl CoA to M-CPTI was completely abolished in the Delta28, Delta39, Delta51, and Delta72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme. Delta18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity malonyl CoA binding similar to the wild type. Replacement of the N-terminal domain of L-CPTI with the N-terminal domain of M-CPTI does not change the malonyl CoA sensitivity of the chimeric L-CPTI, suggesting that the amino acid residues responsible for the differing sensitivity to malonyl CoA are not located in this N-terminal region. These results demonstrate that the N-terminal residues critical for activity and malonyl CoA sensitivity in M-CPTI are different from those of L-CPTI.     

Analysis of keystone enzyme in Agar hydrolysis provides insight into the degradation (of a polysaccharide from) red seaweeds

Agars are abundant polysaccharides from marine red algae, and their chemical structure consists of alternating D-galactose and 3,6-anhydro-L-galactose residues, the latter of which are presumed to make the polymer recalcitrant to degradation by most terrestrial bacteria. Here we study a family 117 glycoside hydrolase (BpGH117) encoded within a recently discovered locus from the human gut bacterium Bacteroides plebeius. Consistent with this locus being involved in agarocolloid degradation, we show that BpGH117 is an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase that removes the 3,6-anhydrogalactose from the non-reducing end of neoagaro-oligosaccharides. A Michaelis complex of BpGH117 with neoagarobiose reveals the distortion of the constrained 3,6-anhydro-L-galactose into a conformation that favors catalysis. Furthermore, this complex, supported by analysis of site-directed mutants, provides evidence for an organization of the active site and positioning of the catalytic residues that are consistent with an inverting mechanism of catalysis and suggests that a histidine residue acts as the general acid. This latter feature differs from the vast majority of glycoside hydrolases, which use a carboxylic acid, highlighting the alternative strategies that enzymes may utilize in catalyzing the cleavage of glycosidic bonds.     

Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin

The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an "editing" enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6-deoxyerythronolide B synthase; AT(L)-ACP(L)). The pikromycin TEII exhibited high K(m) values (>100 microm) with all substrates and no apparent ACP specificity, catalyzing cleavage of methylmalonyl-ACP from both AT(L)-ACP(L) (k(cat)/K(m) 3.3 +/- 1.1 m(-1) s(-1)) and PikAIII (k(cat)/K(m) 2.9 +/- 0.9 m(-1) s(-1)). The TEII exhibited some acyl-group specificity, catalyzing hydrolysis of propionyl (k(cat)/K(m) 15.8 +/- 1.8 m(-1) s(-1)) and butyryl (k(cat)/K(m) 17.5 +/- 2.1 m(-1) s(-1)) derivatives of AT(L)-ACP(L) faster than acetyl (k(cat)/K(m) 4.9 +/- 0.7 m(-1) s(-1)), malonyl (k(cat)/K(m) 3.9 +/- 0.5 m(-1) s(-1)), or methylmalonyl derivatives. PikAIV containing a TEI domain catalyzed cleavage of propionyl derivative of AT(L)-ACP(L) at a dramatically lower rate than TEII. These results provide the first unequivocal in vitro evidence that TEII can hydrolyze acyl-ACP thioesters and a model for the action of TEII in which the enzyme remains primarily dissociated from the polyketide synthase, preferentially removing aberrant acyl-ACP species with long half-lives. The lack of rigorous substrate specificity for TEII may explain the surprising observation that high level expression of the protein in Streptomyces venezuelae leads to significant (>50%) titer decreases.     

Immunopurification and characterization of a 40-kD 1-aminocyclopropane-1-carboxylic acid N-malonyltransferase from mung bean seedling hypocotyls.

1-Aminocyclopropane-1-carboxylic acid (ACC) N-malonyltransferase catalyzes the transfer of the malonyl group from malonyl coenzyme A to ACC to form malonyl ACC. Using partially purified ACC N-malonyltransferase from the hypocotyls of mung bean (Vigna radiata) seedlings, we produced two mouse monoclonal antibodies (1H5 and 2G3) to this enzyme. These antibodies bind to sites other than the active site of the enzyme because monoclonal antibody-bound ACC N-malonyltransferase still exhibits full catalytic activity. A monoclonal antibody column was constructed using 1H5 and protein G Sepharose. The ACC N-malonyltransferase purified from this monoclonal antibody column has a molecular mass of 40 kD, which is different from that reported previously. The enzyme has a higher electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of the reducing agent dithiothreitol. The optimum temperature of this 40-kD ACC N-malonyltransferase is 45 degrees C and the apparent Kms for ACC and malonyl coenzyme A are 66.7 and 40 microns, respectively.     

Irreversible inactivation of chicken liver fatty acid synthetase by its substrates acetyl and malonyl CoA: Effect of temperature and NADP+ on fatty acid and triacetic acid lactone synthesis

Chicken liver fatty acid synthetase is irreversibly inactivated by malonyl CoA and by acetyl and malonyl CoA. Two active forms of the enzyme existing above and below 11.5° are inactivated at different rates. Activities for fatty acid and triacetic acid lactone synthesis are lost at about the same rate and NADP⁺ protects the enzyme against inactivation. Inactivation results from the enhanced covalent binding of malonyl groups in addition to those required for fatty acid synthesis.     

Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases

Modular polyketide biosynthesis can be harnessed to generate rationally designed complex natural products through bioengineering. A detailed understanding of the features that govern transfer and processing of polyketide biosynthetic intermediates is crucial to successfully engineer new polyketide pathways. Previous studies have shown that substrate stereochemistry and protein-protein interactions between polyketide synthase modules are both important factors in this process. Here we investigated the substrate tolerance of different polyketide modules and assessed the relative importance of inter-module chain transfer versus chain elongation activity of some of these modules. By constructing a variety of hybrid modular polyketide synthase systems and assaying their ability to generate polyketide products, it was determined that the substrate tolerance of each individual ketosynthase domain is an important parameter for the successful recombination of polyketide synthase modules. Surprisingly, however, failure by a module to process a candidate substrate was not due to its inability to bind to it. Rather, it appeared to result from a blockage in carbon-carbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzyme-catalyzed decarboxylative condensation reaction.     

Enzymic synthesis of an aromatic ring from acetate units. Partial purification and some properties of flavanone synthase from cell-suspension cultures of Petroselinum hortense.

Flavanone synthase was isolated and purified about 300-fold from fermenter-grown, light-induced cell suspension cultures of Petroselinum hortense. The enzyme catalyzed the formation of the flavanone naringenin from p-coumaroyl-CoA and malonyl-CoA. Trapping experiments with an enzyme preparation, which was free of chalcone isomerase activity, revealed that in fact the flavanone and not the isomeric chalcone was the immediate product of the synthase reaction. Thus the enzyme is not a chalcone synthase as previously assumed. No coafactors were required for flavanone synthase activity. The enzyme was strongly inhibited by the two reaction products naringenin and CoASH, by the antibiotic cerulenin, by acetyl-CoA, and by several compounds reacting with sulfhydryl groups. Optimal enzyme activity was found at pH 8.0, at 30 degrees C, and at an ionic strength of 0.1--0.3 M potassium phosphate. EDTA, Mg2+, Ca2+, or Fe2+ at concentrations of about 0.7 muM did not affect the enzyme activity. Apparent molecular weights of approx. 120 000, 50 000, and 70 000, respectively, were determined for flavanone synthase and two metabolically related enzymes, chalcone isomerase and malonyl-CoA: flavonoid glycoside malonyl transferase. The partially purified flavanone synthase efficiently catalyzed the formation of malonyl pantetheine from malonyl-CoA and pantetheine. This malonyl transferase activity, and a general similarity with the condensation steps involved in the mechanisms of fatty acid and 6-methylsalicylic acid synthesis from "acetate units", are the basis for a hypothetical scheme which is proposed for the sequence of reactions catalyzed by the multifunctional flavanone synthase.     

New experiments of biotin enzymes.

The objects of structural studies on biotin-enzymes were acetyl CoA-carboxylase and pyruvate carboxylase of Saccharomyces cerevisiae and beta-methylcrotonyl CoA-carboxylase and acetyl CoA-carboxylase of Achromobacter IV S. It was found that these enzymes can be arranged in three groups. In the first group, as represented by acetyl CoA-carboxylase of Achromobacter, the active enzyme could be resolved in three types of functional components: (1) the biotin-carboxyl carrier protein, (2) the biotin carboxylase, and (3) the carboxyl transferase. In the second group, as represented by beta-methylcrotonyl CoA-carboxylase from Achromobacter only two types of polypeptides are present. The one carries the biotin carboxylase activity together with the biotin-carboxyl-carrier protein, the other one carries the carboxyl transferase activity. In this third group, as represented by the two enzymes of yeast, all three catalytic functions are incorporated in one multifunctional polypeptide chain. The evolution of the different enzymes is discussed. The animal tissues acetyl CoA-carboxylase is under metabolic control, as known from previous studies. It thus has to be expected that the levels of malonyl CoA in livers of rats in all states of depressed fatty acid synthesis are much lower than under normal conditions because the carboxylation of acetyl CoA is strongly reduced and cannot keep pace with the consumption of malonyl CoA by fatty acid synthetase. A new highly sensitive assay method for malonyl CoA was developed which uses tritiated NADPH and measures the incorporation of radioactivity into the fatty acids formed from malonyl CoA in the presence of purified fatty acid synthetase. The application of this method to liver extracts showed that the level of malonyl CoA which amounts to about 7 nmoles per gram of wet liver drops to less than 10% within a starvation period of 24 hr and even further if the starvation period is extended to 48 hr. A low malonyl CoA concentration is also found in the alloxan diabetic animals and in animals being fed a fatty diet after starvation. On the other hand, feeding a carbohydrate rich diet leads to malonyl CoA levels surpassing the levels found after feeding a balanced diet. These observations reconfirm the concept that fatty acid synthesis is principally regulated by the carboxylation of acetyl CoA.     

In vitro reconstitution and analysis of the chain initiating enzymes of the R1128 polyketide synthase.

Biosynthesis of the carbon chain backbone of the R1128 substances is believed to involve the activity of a ketosynthase/chain length factor (ZhuB/ZhuA), an additional ketosynthase (ZhuH), an acyl transferase (ZhuC), and two acyl carrier proteins (ACPs; ZhuG and ZhuN). A subset of these proteins initiate chain synthesis via decarboxylative condensation between an acetyl-, propionyl-, isobutyryl-, or butyryl-CoA derived primer unit and a malonyl-CoA derived extender unit to yield an acetoacetyl-, beta-ketopentanoyl-, 3-oxo-4-methylpentanoyl-, or beta-ketohexanoyl-ACP product, respectively. To investigate the precise roles of ZhuH, ZhuC, ZhuG, and ZhuN, each protein was expressed in Escherichia coli and purified to homogeneity. Although earlier reports had proposed that ZhuC and its homologues played a role in primer unit selection, direct in vitro analysis of ZhuC showed that it was in fact a malonyl-CoA:ACP malonyltransferase (MAT). The enzyme could catalyze malonyl transfer but not acetyl- or propionyl-transfer onto R1128 ACPs or onto ACPs from other biosynthetic pathways, suggesting that ZhuC has broad substrate specificity with respect to the holo-ACP substrate but is specific for malonyl-CoA. Thus, ZhuC supplies extender units to both the initiating and elongating ketosynthases from this pathway. To interrogate the primer unit specificity of ZhuH, the kinetics of beta-ketoacyl-ACP formation in the presence of various acyl-CoAs and malonyl-ZhuG were measured. Propionyl-CoA and isobutyryl-CoA were the two most preferred substrates of ZhuH, although acetyl-CoA and butyryl-CoA could also be accepted and elongated. This specificity is not only consistent with earlier reports demonstrating that R1128B and R1128C are the major products of the R1128 pathway in vivo, but is also in good agreement with the properties of the ZhuH substrate binding pocket, as deduced from a recently solved crystal structure of the enzyme. Finally, to investigate the molecular logic for the occurrence of not one but two ACP genes within the R1128 gene cluster, the inhibition of ZhuH-catalyzed formation of beta-ketopentanoyl-ACP was quantified in the presence of apo-ZhuG or apo-ZhuN. Both apo-proteins were comparable inhibitors of the ZhuH catalyzed reaction, suggesting that the corresponding apo-proteins can be used interchangeably during chain initiation. Together, these results provide direct biochemical insights into the mechanism of chain initiation of an unusual bacterial aromatic PKS.     

Substrate recognition by β-ketoacyl-ACP synthases

β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.     

Substitution of glutamate-3, valine-19, leucine-23, and serine-24 with alanine in the N-terminal region of human heart muscle carnitine palmitoyltransferase I abolishes malonyl CoA inhibition and binding

The muscle isoform of carnitine palmitoyltransferase I (M-CPTI) is 30- to 100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). We have previously shown that deletion of the first 28 N-terminal amino acid residues in M-CPTI abolished malonyl CoA inhibition and high-affinity binding [Biochemistry 39 (2000) 712-717]. To determine the role of specific residues within the first 28 N-terminal amino acids of human heart M-CPTI on malonyl CoA sensitivity and binding, we constructed a series of substitution mutations and a mutant M-CPTI composed of deletion 18 combined with substitution mutations V19A, L23A, and S24A. All mutants had CPT activity similar to that of the wild type. A change of Glu3 to Ala resulted in a 60-fold decrease in malonyl CoA sensitivity and loss of high-affinity malonyl CoA binding. A change of His5 to Ala in M-CPTI resulted in only a 2-fold decrease in malonyl CoA sensitivity and a significant loss in the low- but not high-affinity malonyl CoA binding. Deletion of the first 18 N-terminal residues combined with substitution mutations V19A, L23A, and S24A resulted in a mutant M-CPTI with an over 140-fold decrease in malonyl CoA sensitivity and a significant loss in both high- and low-affinity malonyl CoA binding. This was further confirmed by a combined four-residue substitution of Glu3, Val19, Leu23, and Ser24 with alanine. Our site-directed mutagenesis studies demonstrate that Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl CoA inhibition and binding, but not for catalysis.