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Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis.

ABSTRACT: Polyketide natural products possess diverse architectures and biological functions and share a subset of biosynthetic steps with fatty acid synthesis. The final transformation catalyzed by both polyketide synthases (PKSs) and fatty acid synthases is most often carried out by a thioesterase (TE). The synthetic versatility of TE domains in fungal nonreducing, iterative PKSs (NR-PKSs) has been shown to extend to Claisen cyclase (CLC) chemistry by catalyzing C-C ring closure reactions as opposed to thioester hydrolysis or O-C/N-C macrocyclization observed in previously reported TE structures. Catalysis of C-C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, but how this activity was acquired has remained a mystery. We report the biochemical and structural analyses of the TE/CLC domain in polyketide synthase A, the multidomain PKS central to the biosynthesis of aflatoxin B(1), a potent environmental carcinogen. Mutagenesis experiments confirm the predicted identity of the catalytic triad and its role in catalyzing the final Claisen-type cyclization to the aflatoxin precursor, norsolorinic acid anthrone. The 1.7 A crystal structure displays an alpha/beta-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber. We propose that a key rotation of the substrate side chain coupled to a protein conformational change from the open to closed form spatially governs substrate positioning and C-C cyclization. The biochemical studies, the 1.7 A crystal structure of the TE/CLC domain, and intermediate modeling afford the first mechanistic insights into this widely distributed C-C bond-forming class of TEs.

SUBMITTER: Korman TP

PROVIDER: S-EPMC2851968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis.

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