Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases.
ABSTRACT: The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.
Project description:Engineering of assembly line polyketide synthases (PKSs) to produce novel bioactive compounds has been a goal for over 20 years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain-domain interactions are evolutionarily optimized and, if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a noncovalent docking domain, or SYNZIP domain, mediated interface. These chimeric systems exhibited non-native domain-domain interactions during intermodular polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in which a noncovalent interface was introduced between the condensing and processing parts of a module, resulting in non-native domain interactions during the extender unit acylation and polyketide chain elongation steps of their catalytic cycles. We show that the natural PKS docking domains can be efficiently substituted with SYNZIP domains and that the newly introduced noncovalent interface between the condensing and processing parts of a module can be harnessed for PKS engineering. Additionally, we established SYNZIP domains as a new tool for engineering PKSs by efficiently bridging non-native interfaces without perturbing PKS activity.
Project description:Individual modules of modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) consist of conserved, covalently linked domains separated by unconserved intervening linker sequences. To better understand the protein-protein and enzyme-substrate interactions in modular catalysis, we have exploited recent structural insights to prepare stand-alone domains of selected DEBS modules. When combined in vitro, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and diketide substrate elongation. When added to a minimal PKS, ketoreductase domains from DEBS modules 1, 2, and 6 showed specificity for the beta-ketoacylthioester substrate, but not for either the ACP domain carrying the polyketide substrate or the KS domain that synthesized the substrate. With insights into catalytic efficiency and specificity of PKS modules, our results provide guidelines for constructing optimal hybrid PKS systems.
Project description:Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the ?-methyl and ?-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed an ~10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed an ~10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules.
Project description:LovF is a highly reducing polyketide synthase (HR-PKS) from the filamentous fungus Aspergillus terreus. LovF synthesizes the alpha-S-methylbutyrate side chain that is subsequently transferred to monacolin J to yield the cholesterol-lowering natural product lovastatin. In the report, we expressed the full length LovF and reconstituted the megasynthase activities in vitro. We confirmed the diketide product of LovF is offloaded from the LovF ACP domain by the dissociated acyltransferase LovD. This represents the first example of acyltransferase-mediated release of polyketide products from fungal PKSs. We determined LovD primarily interacts with the ACP domain of LovF and the protein-protein interactions lead to highly efficient transfer of the diketide product. The catalytic efficiency is enhanced nearly 1 x 10(6)-fold when LovF was used as the acyl carrier instead of N-acetylcysteamine. Reconstitution and characterization of the LovF offloading mechanism provide new insights into the functions of fungal HR-PKS.
Project description:Modularity is a fundamental property of megasynthases such as polyketide synthases (PKSs). In this study, we exploit the close resemblance between PKSs and animal fatty acid synthase (FAS) to re-engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by sequence and structural information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS-like modules as well as bimodular constructs. The novel re-engineered modules resemble all four common types of PKSs and demonstrate that this approach can be a powerful tool to deliver products with higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS-based biosynthetic pathways for custom compound design.
Project description:The pikromyin polyketide synthase (PKS) in Streptomyces venezulae is comprised of a loading module and six extension modules, which generate the corresponding 14-membered macrolactone product. PikAI is a multimodular component of this PKS and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. We have shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains (DDs) from a heterologous modular phoslactomycin PKS are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the S. venezuelae mutant were 50% of that of a S. venezuelae strain expressing the native trimodular PikAI. This observation provides the first demonstration that such separations do not dramatically impact the efficiency of the entire in vivo biosynthetic process. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a tenfold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. These results demonstrate the utility of DDs to manipulate biosynthetic processes catalyzed by modular PKSs and the quest to generate novel polyketide products.
Project description:Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs). The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.
Project description:Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.
Project description:Polyketide synthase (PKS) catalyzes the biosynthesis of polyketides, which are structurally and functionally diverse natural products in microorganisms and plants. Here, we have analyzed available full genome sequences of microscopic and macroscopic algae for the presence of type I PKS genes.Type I PKS genes are present in 15 of 32 analyzed algal species. In chlorophytes, large proteins in the MDa range are predicted in most sequenced species, and PKSs with free-standing acyltransferase domains (trans-AT PKSs) predominate. In a phylogenetic tree, PKS sequences from different algal phyla form clades that are distinct from PKSs from other organisms such as non-photosynthetic protists or cyanobacteria. However, intermixing is observed in some cases, for example polyunsaturated fatty acid (PUFA) and glycolipid synthases of various origins. Close relationships between type I PKS modules from different species or between modules within the same multimodular enzyme were identified, suggesting module duplications during evolution of algal PKSs. In contrast to type I PKSs, nonribosomal peptide synthetases (NRPSs) are relatively rare in algae (occurrence in 7 of 32 species).Our phylogenetic analysis of type I PKSs in algae supports an evolutionary scenario whereby integrated AT domains were displaced to yield trans-AT PKSs. Together with module duplications, the displacement of AT domains may constitute a major mechanism of PKS evolution in algae. This study advances our understanding of the diversity of eukaryotic PKSs and their evolutionary trajectories.
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.