Structural rearrangements of a polyketide synthase module during its catalytic cycle (original) (raw)
Related papers
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.
Faculty of 1000 evaluation for Structure of a modular polyketide synthase
F1000 - Post-publication peer review of the biomedical literature, 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, has an architecture in which successive modules catalyze two-carbon linear extensions and keto group processing reactions on intermediates covalently tethered to carrier domains. We employed electron cryo-microscopy to visualize a full-length module and determine sub-nanometer resolution 3D reconstructions 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 intra-module 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 the structural basis for both intra-module and inter-module substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
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, 2011
Type II polyketides include antibiotics such as tetracycline, and chemotherapeutics such as daunorubicin. Type II polyketides are biosynthesized by the type II polyketide synthase (PKS) that consists of 5-10 stand-alone domains. In many type II PKSs, the type II ketoreductase (KR) specifically reduce the C9-carbonyl group. How the type II KR achieves such a high regiospecificity, and the nature of stereo-specificity, are not well understood. Sequence alignment of KRs led to a hypothesis that a well-conserved 94-XGG-96 motif may be involved in controlling the stereochemistry. The stereo-specificity of single, double and triple mutant combinations of P94L, G95D and G96D were analyzed in vitro and in vivo for the actinorhodin KR (actKR). The P94L mutation is sufficient to change the stereospecificity of actKR. Binary and ternary crystal structures of both wild type and P94L actKR were solved. Together with assay results, docking simulations, and co-crystal structures, a model for stereochemical control is presented herein that elucidates how type II polyketides are introduced into the substrate pocket such that the C9carbonyl can be reduced with high regio-and stereo-specificities. The molecular features of actKR important for regio-and stereo-specificities can potentially be applied to biosynthesize new polyketides via protein engineering that rationally controls polyketide ketoreduction. Streptomycete soil bacteria are one of nature's largest producers of clinically relevant pharmaceuticals (1, 2). A majority of these products belong to the polyketide family, which are used as antibiotic (actinorhodin), anti-cancer (daunorubicin), and immunosuppressive (FK506) agents (3). The wide range of bioactivities of polyketide natural products is due in part to the presence of multiple chiral centers that contribute to their structural diversity. Recently, the importance of developing chiral drugs and their effect on pharmacokinetics have been reviewed (4). Often, the specific bioactivity and biosynthesis of these natural products are dependent on the presence of chiral centers introduced in a controlled and systematic manner. In fact, many natural products lack biological activity in the absence of modifications such as hydroxylations and glycosylations that create chiral centers (5). The importance of chirality for the action and biosynthesis of polyketides therefore presents a new facet that can be controlled to develop new, therapeutic polyketide products. The chemical diversity of aromatic polyketides arises as a result of biosynthesis by polyketide synthase (PKS), which is closely related to fatty acid synthase (FAS). Both FAS
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
Proceedings of the National Academy of Sciences, 2001
As the first structural elucidation of a modular polyketide synthase (PKS) domain, the crystal structure of the macrocycle-forming thioesterase (TE) domain from the 6-deoxyerythronolide B synthase (DEBS) was solved by a combination of multiple isomorphous replacement and multiwavelength anomalous dispersion and refined to an R factor of 24.1% to 2.8-Å resolution. Its overall tertiary architecture belongs to the ␣͞-hydrolase family, with two unusual features unprecedented in this family: a hydrophobic leucine-rich dimer interface and a substrate channel that passes through the entire protein.
Biochemistry, 2004
Aromatic polyketides are a class of natural products that include many pharmaceutically important aromatic compounds. Understanding the structure and function of PKS will provide clues to the molecular basis of polyketide biosynthesis specificity. Polyketide chain reduction by ketoreductase (KR) provides regio-and stereochemical diversity. Two cocrystal structures of actinorhodin polyketide ketoreductase (act KR) were solved to 2.3 Å with either the cofactor NADP + or NADPH bound. The monomer fold is a highly conserved Rossmann fold. Subtle differences between structures of act KR and fatty acid KRs fine-tune the tetramer interface and substrate binding pocket. Comparisons of the NADP +-and NADPH-bound structures indicate that the α6-α7 loop region is highly flexible. The intricate proton-relay network in the active site leads to the proposed catalytic mechanism involving four waters, NADPH, and the active site tetrad Asn114-Ser144-Tyr157-Lys161. Acyl carrier protein and substrate docking models shed light on the molecular basis of KR regio-and stereoselectivity, as well as the differences between aromatic polyketide and fatty acid biosyntheses. Sequence comparison indicates that the above features are highly conserved among aromatic polyketide KRs. The structures of act KR provide an important step toward understanding aromatic PKS and will enhance our ability to design novel aromatic polyketide natural products with different reduction patterns. Nature creates a huge array of natural products that are diverse in their chemical structures and bioactivity. One such example are the polyketides, a large family of natural products that are an extremely rich source of bioactive molecules (1,2). Representative compounds include cholesterol-lowering drugs (such as lovastatin) (3), antibiotics (such as tetracyclines and actinorhodin), and anticancer agents (such as doxorubicin, Figure 1A) (1,2). The biosynthesis of these medically important polyketides is achieved by polyketide synthase (PKS), 1 which synthesizes polyketides in high quantity and yields. Similar to fatty acid synthase (FAS), the PKSs are multifunctional enzymes that catalyze repeated chain elongations followed by optional chain modifications (4). The variation in chain length, choice of chain-building units, and chain modifications leads to the huge diversity among naturally occurring polyketides. Over the past decade, PKSs have been targets of intensive manipulation and analysis via genetic engineering (2,5). These studies have given rise to >100 "unnatural" natural products as well as new technologies for manipulating natural product biosynthesis (6,7). However, this endeavor has been severely hampered by the lack of molecular information about PKS subunits. Structural analyses of PKS subunits will help to answer important questions about polyketide ‡ The atomic coordinates have been deposited in the Protein Data Bank (accession codes 1X7G and 1X7H).
Mechanism of intersubunit ketosynthase-dehydratase interaction in polyketide synthases
Nature chemical biology, 2018
Modular polyketide synthases (PKSs) produce numerous structurally complex natural products that have diverse applications in medicine and agriculture. PKSs typically consist of several multienzyme subunits that utilize structurally defined docking domains (DDs) at their N and C termini to ensure correct assembly into functional multiprotein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-acyltransferase (trans-AT) modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This mechanism involves direct interaction of a largely unstructured docking domain (DD) at the C terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based crosslinking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene footprinting, and mutagenes...