Structure of a modular polyketide synthase (original) (raw)

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...

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:

Biochemical characterization of the minimal domains of an iterative eukaryotic polyketide synthase

The FEBS Journal

Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, b-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and b-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono-to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the b-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs.

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.

The Structure of Docking Domains in Modular Polyketide Synthases

Chemistry & Biology, 2003

that house extender modules (i.e., the regions N-terminal of the KS domain) contain regularities in their University of Cambridge 80 Tennis Court Road amino acid sequence typical of amphipathic parallel ␣-helical coiled coils [10], led to the proposal that these Cambridge CB2 1GA United Kingdom N termini are involved in specific coiled-coil interactions that stabilize PKS homodimeric assemblies . More recent studies have highlighted the potential role of these regions as "linkers" interacting with partner Summary "linker" regions at the extreme C termini of the previous PKS multienzyme. These linker regions are referred to Polyketides from actinomycete bacteria provide the here as "docking domains," given that they adopt a basis for many valuable medicines, so engineering specific three-dimensional fold, as discussed below. genes for their biosynthesis to produce variant mole-Khosla and colleagues have reported that docking docules holds promise for drug discovery. The modular main partners can be substituted by other such partners polyketide synthases are particularly amenable to this without impairing biological function of the PKS [12, approach, because each cycle of chain extension is 13], and that they can also mediate acyl chain transfer catalyzed by a different module of enzymes, and the between some domains and modules that do not normodules are arranged within giant multienzyme submally cooperate with each other . Furthermore, units in the order in which they act. Protein-protein they have proposed that intersubunit protein-protein recinteractions between terminal docking domains of ognition is mediated by interactions between helices [11]. successive multienzymes promote their correct posi-Although the interface between successive multientioning within the assembly line, but because the overzymes likely also involves ACP and KS domains [15, all complex is not stable in vitro, the key interactions 16], it is clear that the intermolecular docking-domain have not been identified. We present here the NMR interaction is central to an understanding of the strucsolution structure of a 120 residue polypeptide repretural basis for discrimination between potential partners senting a typical pair of such domains, fused at their and, therefore, to attempts to improve hybrid PKSs.

New Start and Finish for Complex Polyketide Biosynthesis

Chemistry & Biology, 2004

as well as a loading module for transferring the starter acyl group onto the first KS domain. This modular orga-Biosynthesis nization allows programmed assembly of a defined sequence of starter and extender units, together with controlled processing of each ␤-ketone group. The final The polyketide vicenistatin has significant anticancer product may be cyclized by a thioesterase (TE) to give activity. In the January issue of Chemistry & Biology, a macrolactone.