Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase

Aloesone synthase (ALS) and chalcone synthase (CHS) are plant-specific type III poyketide synthases sharing 62% amino acid sequence identity. ALS selects acetyl-CoA as a starter and carries out six successive condensations with malonyl-CoA to produce a heptaketide aloesone, whereas CHS catalyses condensations of 4-coumaroyl-CoA with three malonyl-CoAs to generate chalcone. In ALS, CHS's Thr197, Gly256, and Ser338, the active site residues lining the initiation/elongation cavity, are uniquely replaced with Ala, Leu, and Thr, respectively. A homology model predicted that the active site architecture of ALS combines a 'horizontally restricting' G256L substitution with a 'downward expanding' T197A replacement relative to CHS. Moreover, ALS has an additional buried pocket that extends into the 'floor' of the active site cavity. The steric modulation thus facilitates ALS to utilize the smaller acetyl-CoA starter while providing adequate volume for the additional polyketide chain extensions. In fact, it was demonstrated that CHS-like point mutations at these positions (A197T, L256G, and T338S) completely abolished the heptaketide producing activity. Instead, A197T mutant yielded a pentaketide, 2,7-dihydroxy-5-methylchromone, while L256G and T338S just afforded a triketide, triacetic acid lactone. In contrast, L256G accepted 4-coumaroyl-CoA as starter to efficiently produce a tetraketide, 4-coumaroyltriacetic acid lactone. These results suggested that Gly256 determines starter substrate selectivity, while Thr197 located at the entrance of the buried pocket controls polyketide chain length. Finally, Ser338 in proximity of the catalytic Cys164 guides the linear polyketide intermediate to extend into the pocket, thus leading to formation of the hepataketide in Rheum palmatum ALS.     

Site-directed mutagenesis of benzalacetone synthase. The role of the Phe215 in plant type III polyketide synthases

Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type III polyketide synthases (PKSs) that share approximately 70% amino acid sequence identity. BAS catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce a diketide benzalacetone, whereas CHS performs sequential condensations with three malonyl-CoA to generate a tetraketide chalcone. A homology model suggested that BAS has the same overall fold as CHS with cavity volume almost as large as that of CHS. One of the most characteristic features is that Rheum palmatum BAS lacks active site Phe-215; the residues 214LF conserved in type III PKSs are uniquely replaced by IL. Our observation that the BAS I214L/L215F mutant exhibited chalcone-forming activity in a pH-dependent manner supported a hypothesis that the absence of Phe-215 in BAS accounts for the interruption of the polyketide chain elongation at the diketide stage. On the other hand, Phe-215 mutants of Scutellaria baicalensis CHS (L214I/F215L, F215W, F215Y, F215S, F215A, F215H, and F215C) afforded increased levels of truncated products; however, none of them generated benzalacetone. These results confirmed the critical role of Phe-215 in the polyketide formation reactions and provided structural basis for understanding the structure-function relationship of the plant type III PKSs.     

Structure function analysis of benzalacetone synthase from Rheum palmatum

Benzalacetone synthase (BAS) is a plant-specific chalcone synthase (CHS) superfamily type III polyketide synthase (PKS) that catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA. The diketide forming activity of Rheum palmatum BAS is attributed to the characteristic substitution of the conserved active-site Phe215 with Leu (numbering in Medicago sativa CHS). To further understand the structure and function of R. palmatum BAS, four site-directed mutants (C197T, C197G, G256L, and S338V) were newly constructed. All the mutants did not change the product pattern, however, the activity was 2-fold increased in S338V, while reduced to half in G256L mutant. On the other hand, the C197 mutants were functionally almost identical to wild-type BAS, excluding the possibility that the second active-site Cys is involved in the enzyme reaction. Instead, homology modeling suggested a possibility that, unlike the case of CHS, BAS utilizes an alternative pocket to lock the coumaroyl moiety for the diketide formation reaction.     

Structure-based engineering of benzalacetone synthase

Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type III polyketide synthases (PKSs), sharing 70% amino acid sequence identity and highly homologous overall protein structures. BAS catalyzes the decarboxylative coupling of 4-coumaroyl-CoA with malonyl-CoA to produce the diketide benzalacetone, whereas CHS produces the tetraketide chalcone by iterative condensations with three molecules of malonyl-CoA, and folding the resulting intermediate into a new aromatic ring system. Recent crystallographic analyses of Rheum palmatum BAS revealed that the characteristic substitution of Thr132 (numbering of Medicago sativa CHS2), a conserved CHS residue lining the active-site cavity, with Leu causes steric contraction of the BAS active-site to produce the diketide, instead of the tetraketide. To test this hypothesis, we constructed a set of R. palmatum BAS site-directed mutants (L132G, L132A, L132S, L132C, L132T, L132F, L132Y, L132W and L132P), and investigated the mechanistic consequences of the point mutations. As a result, the single amino acid substitution L132T restored the chalcone-forming activity in BAS, whereas the Ala, Ser, and Cys substitutions expanded the product chain length to produce 4-coumaroyltriacetic acid lactone (CTAL) after three condensations with malonyl-CoA, but without the formation of the aromatic ring system. Homology modeling suggested that this is probably caused by the restoration of the ‘coumaroyl binding pocket’ in the active-site cavity. These findings provide further insights into the structural details of the catalytic mechanism of the type III PKS enzymes.     

Molecular Cloning,Modeling, and Site-Directed Mutagenesis of Type III Polyketide Synthase from <Emphasis Type="Italic">Sargassum binderi</Emphasis> (Phaeophyta)

Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C₆–C₁₄) to produce tri- and tetraketide pyrones. Mutations at H³³¹ and N³⁶⁴ caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His²²⁷ and Leu³⁶⁶ play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues.     

Mechanism of chalcone synthase. pKa of the catalytic cysteine and the role of the conserved histidine in a plant polyketide synthase

Polyketide synthases (PKS) assemble structurally diverse natural products using a common mechanistic strategy that relies on a cysteine residue to anchor the polyketide during a series of decarboxylative condensation reactions that build the final reaction product. Crystallographic and functional studies of chalcone synthase (CHS), a plant-specific PKS, indicate that a cysteine-histidine pair (Cys(164)-His(303)) forms part of the catalytic machinery. Thiol-specific inactivation and the pH dependence of the malonyl-CoA decarboxylation reaction were used to evaluate the potential interaction between these two residues. Inactivation of CHS by iodoacetamide and iodoacetic acid targets Cys(164) in a pH-dependent manner (pK(a) = 5.50). The acidic pK(a) of Cys(164) suggests that an ionic interaction with His(303) stabilizes the thiolate anion. Consistent with this assertion, substitution of a glutamine for His(303) maintains catalytic activity but shifts the pK(a) of the thiol to 6.61. Although the H303A mutant was catalytically inactive, the pH-dependent incorporation of [(14)C]iodoacetamide into this mutant exhibits a pK(a) = 7.62. Subsequent analysis of the pH dependence of the malonyl-CoA decarboxylation reaction catalyzed by wild-type CHS and the H303Q and C164A mutants also supports the presence of an ion pair at the CHS active site. Structural and sequence conservation of a cysteine-histidine pair in the active sites of other PKS implies that a thiolate-imidazolium ion pair plays a central role in polyketide biosynthesis.     

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.     

Enzymatic formation of long-chain polyketide pyrones by plant type III polyketide synthases

Recombinant chalcone synthase (CHS) from Scutellaria baicalensis and stilbene synthase (STS) from Arachis hypogaea accepted CoA esters of long-chain fatty acid (CHS up to the C12 ester, while STS up to the C14 ester) as a starter substrate, and carried out sequential condensations with malonyl-CoA, leading to formation of triketide and tetraketide alpha-pyrones. Interestingly, the C6, C8, and C10 esters were kinetically favored by the enzymes over the physiological starter substrate; the kcat/KM values were 1.2- to 1.9-fold higher than that of p-coumaroyl-CoA. The catalytic diversities of the enzymes provided further mechanistic insights into the type III PKS reactions, and suggested involvement of the CHS-superfamily enzymes in the biosynthesis of long-chain alkyl polyphenols such as urushiol and ginkgolic acid in plants.     

Enzymatic formation of an aromatic dodecaketide by engineered plant polyketide synthase

Octaketide synthase (OKS) from Aloe arborescens is a plant-specific type III polyketide synthase (PKS) that catalyzes iterative condensations of eight molecules of malonyl-CoA to produce the C₁₆ aromatic octaketides SEK4 and SEK4b. On the basis of the crystal structures of OKS, the F66L/N222G double mutant was constructed and shown to produce an unnatural dodecaketide TW95a by sequential condensations of 12 molecules of malonyl-CoA. The C₂₄ naphthophenone TW95a is a product of the minimal type II PKS (whiE from Streptomyces coelicolor), and is structurally related to the C₂₀ decaketide benzophenone SEK15, the product of the OKS N222G point mutant. The C₂₄ dodecaketide naphthophenone TW95a is the first and the longest polyketide scaffold generated by a structurally simple type III PKS. A homology model predicted that the active-site cavity volume of the F66L/N222G mutant is increased to 748 Å³, from 652 Å³ of the wild-type OKS. The structure-based engineering thus greatly expanded the catalytic repertoire of the simple type III PKS to further produce larger and more complex polyketide molecules.     

Cloning and characterization of a type III polyketide synthase from Aspergillus niger

Type III polyketide synthases (PKSs) are the condensing enzymes that catalyze the formation of a myriad of aromatic polyketides in plant, bacteria, and fungi. Here we report the cloning and characterization of a putative type III PKS from Aspergillusniger, AnPKS. This enzyme catalyzes the synthesis of alkyl pyrones from C2 to C18 starter CoA thioesters with malonyl-CoA as an extender CoA through decaboxylative condensation and cyclization. It displays broad substrate specificity toward fatty acyl-CoA starters to yield triketide and tetraketide pyrones, with benzoyl-CoA as the most preferred starter. The optimal temperature and pH of AnPKS are 50°C and 8, respectively. Under optimal conditions, the enzyme shows the highest catalytic efficiency (k(cat)/K(m)) of 7.4×10(5)s(-1)M(-1) toward benzoyl-CoA. Homology modeling and site-directed mutagenesis were used to probe the molecular basis of its substrate specificity. This study should open doors for further engineering of AnPKS as a biocatalyst for synthesis of value-added polyketides.     

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.     

Biosynthesis of Aliphatic Polyketides by Type III Polyketide Synthase and Methyltransferase in Bacillus subtilis

Type III polyketide synthases (PKSs) synthesize a variety of aromatic polyketides in plants, fungi, and bacteria. The bacterial genome projects predicted that probable type III PKS genes are distributed in a wide variety of gram-positive and -negative bacteria. The gram-positive model microorganism Bacillus subtilis contained the bcsA-ypbQ operon, which appeared to encode a type III PKS and a methyltransferase, respectively. Here, we report the characterization of bcsA (renamed bpsA, for Bacillus pyrone synthase, on the basis of its function) and ypbQ, which are involved in the biosynthesis of aliphatic polyketides. In vivo analysis demonstrated that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones from long-chain fatty acyl-coenzyme A (CoA) thioesters as starter substrates and malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ thus was named BpsB because of its functional relatedness to BpsA. In vitro analysis with histidine-tagged BpsA revealed that it used broad starter substrates and produced not only triketide pyrones but also tetraketide pyrones and alkylresorcinols. Although the aliphatic polyketides were expected to localize in the membrane and play some role in modulating the rigidity and properties of the membrane, no detectable phenotypic changes were observed for a B. subtilis mutant containing a whole deletion of the bpsA-bpsB operon.Type III polyketide synthases (PKSs), represented by a plant chalcone synthase (CHS), are the condensing enzymes that catalyze the synthesis of aromatic polyketides in plants, fungi, and bacteria (2). CHS catalyzes the decarboxylative condensation of p-coumaroyl-coenzyme A (p-coumaroyl-CoA), called a starter substrate, with three malonyl-CoAs, called extender substrates, and synthesizing a tetraketide intermediate. The synthesized tetraketide intermediate was cyclized and aromatized by CHS and resulted in naringenin chalcone. Like CHS, most of the type III PKSs catalyze the condensation of a starter substrate with several molecules of an extender substrate and cyclization. There are many type III PKSs that differ in these specificities.Until recently, type III PKSs were discovered only from plants. In 1999, the first bacterial type III PKS, RppA, was discovered. RppA catalyzes the condensation of five malonyl-CoAs to synthesize 1,3,6,8-tetrahydroxynaphthalene, which is a precursor of hexahydroxyperylenequinone melanin in the actinomycete Streptomyces griseus (4). Since then, the genome projects of various bacteria have revealed that type III PKSs are widely distributed in a variety of bacteria. For example, ArsB and ArsC, both of which are type III PKSs in Azotobacter vinelandii, catalyze the synthesis of alkylresorcinols and alkylpyrones, respectively, which are essential for encystment as the major lipids in the cyst membrane (5). In S. griseus, the srs operon consisting of srsA, srsB, and srsC is responsible for the synthesis of methylated phenolic lipids derived from alkylresorcinols and alkylpyrones (6). The function of each of the operon members is that SrsA is a type III PKS responsible for the synthesis of phenolic lipids alkylresorcinol and alkylpyrones, SrsB is a methyltransferase acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and SrsC is a hydroxylase acting on the alkylresorcinol methyl ethers. The phenolic lipids synthesized by the Srs enzymes confer resistance to β-lactam antibiotics (6). Therefore, it is suggested that phenolic lipids play an important role as minor components in the biological membrane in various bacteria. In fact, srsAB- and srsABC-like operons are distributed widely in both gram-positive and -negative bacteria (see Fig. S1 in the supplemental material). However, most of these type III PKSs have not been characterized.Bacillus subtilis is one of the best-characterized gram-positive bacteria. BcsA, which stands for bacterial chalcone synthase, was annotated as a homologue of type III PKS in B. subtilis (3). As described in this paper, however, this annotation needs correction. We renamed the gene bpsA (for Bacillus pyrone synthase). Moreover, the functional unknown gene ypbQ is located next to bpsA. YpbQ, consisting of 168 amino acid residues, contained an isoprenylcysteine carboxyl methyltransferase (ICMT) domain of the ICMT family members, which are unique membrane proteins that are involved in the posttranslational modification of oncogenic proteins (23). Therefore, the bpsA and ypbQ genes were predicted to form an operon, just like srsA and srsB in the srs operon in S. griseus. We therefore named ypbQ, a thus-far functionally unknown gene, bpsB.In this study, we characterized the functions of BpsA and BpsB by in vivo and in vitro experiments. The in vivo experiments revealed that the overexpression of bpsA in B. subtilis led to the production of triketide pyrones, and the co-overexpression of bpsA and bpsB led to the production of triketide pyrone methyl ethers. The in vitro analysis showed that BpsA produced triketide pyrones and a small amount of tetraketide pyrones and tetraketide resorcinols from long-chain fatty acyl CoA thioesters as starter substrates and malonyl-CoA as an extender substrate. Therefore, BpsA is a type III PKS that is responsible for the synthesis of alkylpyrones, and BpsB is a methyltransferase that acts on the alkylpyrones to yield alkylpyrone methyl ethers. BpsB is the first enzyme found to methylate alkylpyrones. Furthermore, we attempted to analyze the biological function of the aliphatic polyketides by disrupting the bpsA and bpsB genes, but no distinct phenotypic changes were detected under laboratory conditions.     

Mutational analysis of conserved outer sphere arginine residues of chalcone synthase

Chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, catalyses sequential decarboxylative condensations of p-coumaroyl-CoA with three malonyl-CoA molecules and cyclizes the resulting tetraketide intermediate to produce chalcone. Phenylglyoxal, an Arg selective reagent, was found to inactivate the enzyme, although no Arg is found at the active site. Conserved, non-active site Arg residues of CHS were individually mutated and the results were discussed in the context of the 3D structure of CHS. Arg199 and Arg350 were shown to provide important interactions to maintain the structural integrity and foldability of the enzyme. Arg68, Arg172 and Arg328 interact with highly conserved Gln33/Phe215, Glu380 and Asp311/Glu314, respectively, thus helping position the catalytic Cys-His-Asn triad and the (372)GFGPG loop in correct topology at the active site. In particular, a mutation of Arg172 resulted in selective impairment in the cyclization activities of CHS and stilbene synthase, a related enzyme that catalyses a different cyclization of the same tetraketide intermediate. These Arg residues and their interactions are well conserved in other enzymes of the CHS superfamily, suggesting that they may serve similar functions in other enzymes. Mutations of Arg68 and Arg328 had been found in mutant plants that showed impaired CHS activity.     

LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B encode hydroxyalkyl α-pyrone synthases required for pollen development and sporopollenin biosynthesis in Arabidopsis thaliana

Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.     

Biosynthesis of biphenyls and benzophenones – Evolution of benzoic acid-specific type III polyketide synthases in plants

Type III polyketide synthases (PKSs) generate a diverse array of secondary metabolites by varying the starter substrate, the number of condensation reactions, and the mechanism of ring closure. Among the starter substrates used, benzoyl-CoA is a rare starter molecule. Biphenyl synthase (BIS) and benzophenone synthase (BPS) catalyze the formation of identical linear tetraketide intermediates from benzoyl-CoA and three molecules of malonyl-CoA but use alternative intramolecular cyclization reactions to form 3,5-dihydroxybiphenyl and 2,4,6-trihydroxybenzophenone, respectively. In a phylogenetic tree, BIS and BPS group together closely, indicating that they arise from a relatively recent functional diversification of a common ancestral gene. The functionally diverse PKSs, which include BIS and BPS, and the ubiquitously distributed chalcone synthases (CHSs) form separate clusters, which originate from a gene duplication event prior to the speciation of the angiosperms. BIS is the key enzyme of biphenyl metabolism. Biphenyls and the related dibenzofurans are the phytoalexins of the Maloideae. This subfamily of the Rosaceae includes a number of economically important fruit trees, such as apple and pear. When incubated with ortho-hydroxybenzoyl (salicoyl)-CoA, BIS catalyzes a single decarboxylative condensation with malonyl-CoA to form 4-hydroxycoumarin. A well-known anticoagulant derivative of this enzymatic product is dicoumarol. Elicitor-treated cell cultures of Sorbus aucuparia also formed 4-hydroxycoumarin when fed with the N-acetylcysteamine thioester of salicylic acid (salicoyl-NAC). BPS is the key enzyme of benzophenone metabolism. Polyprenylated benzophenone derivatives with bridged polycyclic skeletons are widely distributed in the Clusiaceae (Guttiferae). Xanthones are regioselectively cyclized benzophenone derivatives. BPS was converted into a functional phenylpyrone synthase (PPS) by a single amino acid substitution in the initiation/elongation cavity. The functional behavior of this Thr135Leu mutant was rationalized by homology modeling. The intermediate triketide may be redirected into a smaller pocket in the active site cavity, resulting in phenylpyrone formation by lactonization.     

Mechanistic analysis of a type II polyketide synthase. Role of conserved residues in the beta-ketoacyl synthase-chain length factor heterodimer

Type II polyketide synthases (PKSs) are a family of multienzyme systems that catalyze the biosynthesis of polyfunctional aromatic natural products such as actinorhodin, frenolicin, tetracenomycin, and doxorubicin. A central component in each of these systems is the beta-ketoacyl synthase-chain length factor (KS-CLF) heterodimer. In the presence of an acyl carrier protein (ACP) and a malonyl-CoA:ACP malonyl transferase (MAT), this enzyme synthesizes a polyketide chain of defined length from malonyl-CoA. We have investigated the role of the actinorhodin KS-CLF in priming, elongation, and termination of its octaketide product by subjecting the wild-type enzyme and selected mutants to assays that probe key steps in the overall catalytic cycle. Under conditions reflecting steady-state turnover of the PKS, a unique acyl-ACP intermediate is detected that carries a long, possibly full-length, acyl chain. This species cannot be synthesized by the C169S, H309A, K341A, and H346A mutants of the KS, all of which are blocked in early steps in the PKS catalytic cycle. These four residues are universally conserved in all known KSs. Malonyl-ACP alone is sufficient for kinetically and stoichiometrically efficient synthesis of polyketides by the wild-type KS-CLF, but not by heterodimers that carry the mutations listed above. Among these mutants, C169S is an efficient decarboxylase of malonyl-ACP, but the H309A, K341A, and H346A mutants are unable to catalyze decarboxylation. Transfer of label from [(14)C]malonyl-ACP to the nucleophile at position 169 in the KS can be detected for the wild-type enzyme and for the C169S and K341A mutants, but not for the H309A mutant and only very weakly for the H346A mutant. A model is proposed for decarboxylative priming and extension of a polyketide chain by the KS, where C169 and H346 form a catalytic dyad for acyl chain attachment, H309 positions the malonyl-ACP in the active site and supports carbanion formation by interacting with the thioester carbonyl, and K341 enhances the rate of malonyl-ACP decarboxylation via electrostatic interaction. Our data also suggest that the ACP and the KS dissociate after each C-C bond forming event, and that the newly extended acyl chain is transferred back from the ACP pantetheine to the KS cysteine before dissociation can occur. Chain termination is most likely the rate-limiting step in polyketide biosynthesis. Within the act CLF, neither the universally conserved S145 residue nor Q171, which aligns with the active site cysteine of the ketosynthase, is essential for PKS activity. The results described here provide a basis for a better understanding of the catalytic cycle of type II PKSs and fatty acid synthases.     

Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase

Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ by KSQ, a ketosynthase-like decarboxylase domain. Consistent with this, the acyltransferase (AT) domains of all KSQ-containing loading modules are shown here to contain a key arginine residue at their active site. Site-specific replacement of this arginine residue in the oleandomycin (ole) loading AT domain effectively abolished AT activity, consistent with its importance for catalysis. Substitution of the ole PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery) loading module gave polyketide products almost wholly either acetate derived or propionate derived, respectively, instead of the mixture found normally. An authentic extension module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately when engineered in the place of the ole loading AT domain, and gave rise to substantial amounts of C13-methylerythromycins, as predicted. The role of direct acylation of the ketosynthase domain of ex-tension module 1 in chain initiation was confirmed by demonstrating that a mutant of the triketide synthase DEBS1-TE, in which the 4'-phosphopante-theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro-ducts in detectable amounts.     

Characterization of the substrate specificity of PhlD, a type III polyketide synthase from Pseudomonas fluorescens

PhlD, a type III polyketide synthase from Pseudomonas fluorescens, catalyzes the synthesis of phloroglucinol from three molecules of malonyl-CoA. Kinetic analysis by direct measurement of the appearance of the CoASH product (k(cat) = 24 +/- 4 min(-1) and Km = 13 +/- 1 microM) gave a k(cat) value more than an order of magnitude higher than that of any other known type III polyketide synthase. PhlD exhibits broad substrate specificity, accepting C4-C12 aliphatic acyl-CoAs and phenylacetyl-CoA as the starters to form C6-polyoxoalkylated alpha-pyrones from sequential condensation with malonyl-CoA. Interestingly, when primed with long chain acyl-CoAs, PhlD catalyzed extra polyketide elongation to form up to heptaketide products. A homology structural model of PhlD showed the presence of a buried tunnel extending out from the active site to assist the binding of long chain acyl-CoAs. To probe the structural basis for the unusual ability of PhlD to accept long chain acyl-CoAs, both site-directed mutagenesis and saturation mutagenesis were carried out on key residues lining the tunnel. Three mutations, M21I, H24V, and L59M, were found to significantly reduce the reactivity of PhlD with lauroyl-CoA while still retaining its physiological activity to synthesize phloroglucinol. Our homology modeling and mutational studies indicated that even subtle changes in the tunnel volume could affect the ability of PhlD to accept long chain acyl-CoAs. This suggested novel strategies for combinatorial biosynthesis of unnatural pharmaceutically important polyketides.     

Structural Basis for Cyclization Specificity of Two Azotobacter Type III Polyketide Synthases: A SINGLE AMINO ACID SUBSTITUTION REVERSES THEIR CYCLIZATION SPECIFICITY*

Type III polyketide synthases (PKSs) show diverse cyclization specificity. We previously characterized two Azotobacter type III PKSs (ArsB and ArsC) with different cyclization specificity. ArsB and ArsC, which share a high sequence identity (71%), produce alkylresorcinols and alkylpyrones through aldol condensation and lactonization of the same polyketomethylene intermediate, respectively. Here we identified a key amino acid residue for the cyclization specificity of each enzyme by site-directed mutagenesis. Trp-281 of ArsB corresponded to Gly-284 of ArsC in the amino acid sequence alignment. The ArsB W281G mutant synthesized alkylpyrone but not alkylresorcinol. In contrast, the ArsC G284W mutant synthesized alkylresorcinol with a small amount of alkylpyrone. These results indicate that this amino acid residue (Trp-281 of ArsB or Gly-284 of ArsC) should occupy a critical position for the cyclization specificity of each enzyme. We then determined crystal structures of the wild-type and G284W ArsC proteins at resolutions of 1.76 and 1.99 Å, respectively. Comparison of these two ArsC structures indicates that the G284W substitution brings a steric wall to the active site cavity, resulting in a significant reduction of the cavity volume. We postulate that the polyketomethylene intermediate can be folded to a suitable form for aldol condensation only in such a relatively narrow cavity of ArsC G284W (and presumably ArsB). This is the first report on the alteration of cyclization specificity from lactonization to aldol condensation for a type III PKS. The ArsC G284W structure is significant as it is the first reported structure of a microbial resorcinol synthase.