The chemical scaffolds of numerous therapeutics are polyketide natural products, many formed by bacterial modular polyketide synthases (PKS). The large and flexible dimeric PKS modules have distinct ...extension and reducing regions. Structures are known for all individual enzyme domains and several extension regions. Here, we report the structure of the full reducing region from a modular PKS, the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains of module 5 of the juvenimicin PKS. The modular PKS-reducing region has a different architecture than the homologous fatty acid synthase (FAS) and iterative PKS systems in its arrangement of domains and dimer interface. The structure reveals a critical role for linker peptides in the domain interfaces, leading to discovery of key differences in KR domains dependent on module composition. Finally, our studies provide insight into the mechanism underlying modular PKS intermediate shuttling by carrier protein (ACP) domains.
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•Modular polyketide synthase reducing regions diverge from iterative megasynthases•Interdomain linkers form “hydrophobic glue” in domain interfaces•KR domains vary depending on the presence or absence of same-module DH domains•Fully reducing PKS modules may function asynchronously
McCullough et al. characterize the architecture of JuvEIII DH-ER-KR, the fully reducing region of module 5 in the juvenimicin polyketide synthase. The structure illustrates the divergence of modular PKS from iterative megasynthase homologs including metazoan fatty acid synthase and reveals structural elements common to PKS modules with fully reducing tridomains.
Modular polyketide synthases (PKS) make novel natural products through a series of preprogrammed chemical steps catalyzed by an assembly line of multidomain modules. Each assembly-line step involves ...unique extension and modification reactions, resulting in tremendous diversity of polyketide products. Dehydratase domains catalyze formation of an α,β-double bond in the nascent polyketide intermediate. We present crystal structures of the four dehydratase domains from the curacin A PKS. The catalytic residues and substrate binding site reside in a tunnel within a single monomer. The positions of the catalytic residues and shape of the substrate tunnel explain how chirality of the substrate hydroxyl group may determine the configuration of the product double bond. Access to the active site may require opening the substrate tunnel, forming an open trench. The arrangement of monomers within the dimer is consistent among PKS dehydratases and differs from that seen in the related mammalian fatty acid synthases.
► Active site location at sharp bend within a tunnel explains how substrate stereochemistry can determine product conformation ► Access to active site is likely mediated by movement of specific structural elements, opening the substrate tunnel to accommodate bulky and/or inflexible substrates ► Re-annotation of Curacin A biosynthetic pathway suggests dehydration reactions may occur across module boundaries ► Orientation of dehydratase monomers in PKS modules differs from that seen for FAS modules but is nonetheless consistent with overall shared architecture of PKS and FAS modules.
Polyketides are a class of biologically active microbial and plant-derived metabolites that possess a high degree of structural and functional diversity and include many human therapeutics, among ...them anti-infective and anti-cancer drugs, growth promoters and anti-parasitic agents. The macrolide antibiotics, characterized by a glycoside-linked macrolactone, constitute an important class of polyketides, including erythromycin and the natural ketolide anti-infective agent pikromycin. Here we describe new mechanistic details of macrolactone ring formation catalyzed by the pikromycin polyketide synthase thioesterase domain from Streptomyces venezuelae. A pentaketide phosphonate mimic of the final pikromycin linear chain-elongation intermediate was synthesized and shown to be an active site affinity label. The crystal structures of the affinity-labeled enzyme and of a 12-membered-ring macrolactone product complex suggest a mechanism for cyclization in which a hydrophilic barrier in the enzyme and structural restraints of the substrate induce a curled conformation to direct macrolactone ring formation.
A narrow tunnel: Biochemical and structural analysis of the tautomycetin thioesterase (TE) has provided the first high‐resolution structure of a linear‐chain‐terminating TE in polyketide ...biosynthesis, showing the enzyme to be stereoselective with a constrained substrate chamber relative to macrolactone‐forming thioesterases.
Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A ...(CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 Å germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.
Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6′-deoxyallose substituent occurs in a ...stepwise manner first at the 2′- and then the 3′-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C–H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3′-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2′-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4′-specific homologue, active site residues were identified that correlate with the 3′ or 4′ specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.
Polyketides are a diverse class of natural products having important clinical properties, including antibiotic, immunosuppressive and anticancer activities. They are biosynthesized by polyketide ...synthases (PKSs), which are modular, multienzyme complexes that sequentially condense simple carboxylic acid derivatives. The final reaction in many PKSs involves thioesterase-catalyzed cyclization of linear chain elongation intermediates. As the substrate in PKSs is presented by a tethered acyl carrier protein, introduction of substrate by diffusion is problematic, and no substrate-bound type I PKS domain structure has been reported so far. We describe the chemical synthesis of polyketide-based affinity labels that covalently modify the active site serine of excised pikromycin thioesterase from Streptomyces venezuelae. Crystal structures reported here of the affinity label-pikromycin thioesterase adducts provide important mechanistic insights. These results suggest that affinity labels can be valuable tools for understanding the mechanisms of individual steps within multifunctional PKSs and for directing rational engineering of PKS domains for combinatorial biosynthesis.
O-linked methylation of sugar substituents is a common modification in the biosynthesis of many natural products and is catalyzed by multiple families of
S-adenosyl-
l-methionine (SAM or ...AdoMet)-dependent methyltransferases (MTs). Mycinamicins, potent antibiotics from
Micromonospora griseorubida, can be methylated at two positions on a 6-deoxyallose substituent. The first methylation is catalyzed by MycE, a SAM- and metal-dependent MT. Crystal structures were determined for MycE bound to the product
S-adenosyl-
l-homocysteine (AdoHcy) and magnesium, both with and without the natural substrate mycinamicin VI. This represents the first structure of a natural product sugar MT in complex with its natural substrate. MycE is a tetramer of a two-domain polypeptide, comprising a C-terminal catalytic MT domain and an N-terminal auxiliary domain, which is important for quaternary assembly and for substrate binding. The symmetric MycE tetramer has a novel MT organization in which each of the four active sites is formed at the junction of three monomers within the tetramer. The active-site structure supports a mechanism in which a conserved histidine acts as a general base, and the metal ion helps to position the methyl acceptor and to stabilize a hydroxylate intermediate. A conserved tyrosine is suggested to support activity through interactions with the transferred methyl group from the SAM methyl donor. The structure of the free enzyme reveals a dramatic order–disorder transition in the active site relative to the
S-adenosyl-
l-homocysteine complexes, suggesting a mechanism for product/substrate exchange through concerted movement of five loops and the polypeptide C-terminus.
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Recent research of the Agricultural Research Service of USDA on the use of natural products to manage pests is summarized. Studies of the use of both phytochemicals and diatomaceous earth to manage ...insect pests are discussed. Chemically characterized compounds, such as a saponin from pepper (Capsicum frutescens L), benzaldehyde, chitosan and 2‐deoxy‐D‐glucose are being studied as natural fungicides. Resin glycosides for pathogen resistance in sweet potato and residues of semi‐tropical leguminous plants for nematode control are also under investigation. Bioassay‐guided isolation of compounds with potential use as herbicides or herbicide leads is underway at several locations. New natural phytotoxin molecular target sites (asparagine synthetase and fructose‐1,6‐bisphosphate aldolase) have been discovered. Weed control in sweet potato and rice by allelopathy is under investigation. Molecular approaches to enhance allelopathy in sorghum are also being undertaken. The genes for polyketide synthases involved in production of pesticidal polyketide compounds in fungi are found to provide clues for pesticide discovery. Gene expression profiles in response to fungicides and herbicides are being generated as tools to understand more fully the mode of action and to rapidly determine the molecular target site of new, natural fungicides and herbicides. Published in 2003 for SCI by John Wiley & Sons, Ltd.
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