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Structure of a modular polyketide synthase
Nature, 2014
Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyse two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to determine sub-nanometre-resolution threedimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems.
Structural rearrangements of a polyketide synthase module during its catalytic cycle
Nature, 2014
The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynth...
Insights into the stereospecificity of ketoreduction in a modular polyketide synthase
Organic & Biomolecular Chemistry, 2011
Ketoreductase enzymes are responsible for the generation of hydroxyl stereocentres during the biosynthesis of complex polyketide natural products. Previous studies of isolated polyketide ketoreductases have shown that the stereospecificity of ketoreduction can be switched by mutagenesis of selected active site amino acids. We show here that in the context of the intact polyketide synthase multienzyme the same changes do not alter the stereochemical outcome in the same way. These findings point towards additional factors that govern ketoreductase stereospecificity on intact multienzymes in vivo. Modular polyketide synthases (PKSs) are multienzyme assembly lines responsible for the biosynthesis of diverse complex polyketide natural products including the macrolide antibiotic erythromycin A, the anticancer epothilones, and the immunosuppressant rapamycin. 1-4 Amongst these enzymes, the 6deoxyerythronolide B synthase (DEBS), which produces the aglycone of erythromycin A, has undoubtedly been the most intensively studied. Type I modular PKSs such as DEBS consist of multiple catalytic domains, distributed over several multienzyme polypeptides, that assemble a polyketide chain by successive head-to-tail condensation of acyl-CoA esters, with the intermediate species remaining covalently bound to the enzyme. 1, 3 In contrast to the type I multienzyme fatty acid synthases (FASs), where multiple cycles of chain elongation proceed by iterative use of a single set of enzymes, type I modular PKSs use distinct sets of domains, or 'modules', to catalyze one round of 65
A Model of Structure and Catalysis for Ketoreductase Domains in Modular Polyketide Synthases
Biochemistry, 2003
A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr f Phe, Ser f Ala, and Lys f Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces liVidans for in vivo analysis. In this case, the Tyr f Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser f Ala and Lys f Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immoblized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.
Biochemistry, 1996
Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex and medicinally important natural products. These large multifunctional enzymes are organized into "modules", where each module contains active sites homologous to those of higher eucaryotic fatty acid synthases (FASs). Like FASs, modular PKSs are known to be dimers. Here we provide functional evidence for the existence of two catalytically independent clusters of active sites within a modular PKS. In three bimodular derivatives of DEBS, the ketosynthase domain of module 1 (KS-1) or module 2 (KS-2) or the acyl carrier protein domain of module 2 (ACP-2) was inactivated via site-directed mutagenesis. As expected, the purified proteins were unable to catalyze polyketide synthesis (although the KS-1 mutant could convert a diketide thioester into the predicted triketide lactone). Remarkably however, the KS-1/KS-2 and the KS-2/ACP-2 mutant pairs could efficiently complement each other and catalyze polyketide formation. In contrast, the KS-1 and ACP-2 mutants did not complement each other. On the basis of these and other results, a model is proposed in which the individual modules of a PKS dimer form head-to-tail homodimers, thereby generating two equivalent and independent clusters of active sites for polyketide biosynthesis. Specifically, each subunit contributes half of the KS and ACP domains in each cluster. A similar complementation approach should also be useful in dissecting the organization of the remaining types of active sites within this family of multienzyme assemblies. Finally, blocked systems, such as the KS-1 mutant described here, present a new strategy for the noncompetitive conversion of unnatural substrates into polyketides by modular PKSs.
An Oxetane-Based Polyketide Surrogate To Probe Substrate Binding in a Polyketide Synthase
Journal of the American Chemical Society, 2018
Polyketides are a large class of bioactive natural products with a wide range of structures and functions. Polyketides are biosynthesized by large, multidomain enzyme complexes termed polyketide synthases (PKSs). One of the primary challenges when studying PKSs is the high reactivity of their poly-β-ketone substrates. This has hampered structural and mechanistic characterization of PKS-polyketide complexes, and, as a result, little is known about how PKSs position the unstable substrates for proper catalysis while displaying high levels of regio- and stereospecificity. As a first step toward a general plan to use oxetanes as carbonyl isosteres to broadly interrogate PKS chemistry, we describe the development and application of an oxetane-based PKS substrate mimic. This enabled the first structural determination of the acyl-enzyme intermediate of a ketosynthase (KS) in complex with an inert extender unit mimic. The crystal structure, in combination with molecular dynamics simulations...
Proceedings of the National Academy of Sciences of the United States of America, 2017
Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsible for controlling the aldol cyclizations of poly-β-ketone intermediates assembled during the catalytic cycle. Our ability to understand the high regioselective control that PT domains exert is hindered by the inaccessibility of intrinsically unstable poly-β-ketones for in vitro studies. We describe here the crystallographic application of "atom replacement" mimetics in which isoxazole rings linked by thioethers mimic the alternating sites of carbonyls in the poly-β-ketone intermediates. We report the 1.8-Å cocrystal structure of the PksA PT domain from aflatoxin biosynthesis with a heptaketide mimetic tethered to a stably modified 4'-phosphopantetheine, which provides important empirical evidence for a previously proposed mechanism of PT-catalyzed cyclization. Key observations support the proposed deprotonation at C4 of the nascent polyketide by the catalytic His1345 and ...