C-O Bond Formation by Polyketide Synthases (original) (raw)
Related papers
An artificial pathway for polyketide biosynthesis
Nature Catalysis, 2020
Polyketide synthases are multi-domain enzymes that catalyse the construction of many bioactive natural products. Now, some of the inefficiencies and limitations of these systems have been solved by designing an artificial pathway for carbon-carbon bond formation via iterative rounds of non-decarboxylative thio-Claisen reactions. Anuran k. Gayen, lindsay nichols and Gavin J. Williams P olyketides are a class of natural products with diverse and potent biological activities, owing to their structural complexity and chemical diversity. Notable examples of polyketides include lovastatin (anticholesterol), doxorubicin (anticancer), ivermectin (antiparasitic), and erythromycin A (macrolide antibiotic). All polyketides are assembled via the repeated decarboxylative thio-Claisen condensation between malonyl-coenzyme A (malonyl-CoA) derivatives catalysed by enzyme machinery called polyketide synthases (PKSs). Type I PKSs are multi-modular mega-enzyme complexes, type II PKSs are a collection of cooperative enzymes, and type III PKSs iteratively catalyse chain elongation via ketosynthase domains. Though they differ in complexity and molecular organization, the three PKS types share similar substrate scopes, which largely consist of malonyl-and methylmalonyl-CoA extender units and simple acyl-CoA starter units, such as acetyl-, propionyl-, and isobutyryl-CoA. Site-selective modular control of oxidation levels by PKSs facilitates structural diversity in the polyketide scaffold that contributes to their biological activity 1. In spite of the proven ability to leverage PKSs for the production of blockbuster polyketide drugs, polyketide biosynthesis suffers from slow reaction kinetics, low energy efficiency, and poor carbon economy-only two out of three extender unit carbons incorporate into the polyketide backbone with loss of CO 2. In addition, the formation of the commonly utilized malonyl-CoA extender unit from acetyl-CoA, ATP and bicarbonate by acetyl-CoA carboxylase (ACC) is highly regulated and a likely rate-limiting step 2. While type II and III PKSs can be less complicated than their type I counterparts, they often do not express well in tractable heterologous hosts such as Escherichia coli (E. coli) and access to substrates is susceptible to competition with primary metabolism. Now, writing in Nature Catalysis, Gonzalez and colleagues report the
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.
ACS synthetic biology, 2016
Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed the segments of AT domains and associated linkers in AT exchanges in vitro and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimi...
A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis
Nature Chemical Biology, 2013
Type I PKSs often utilise programmed β-branching, via enzymes of an "HMG-CoA synthase (HCS) cassette", to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules. Polyketide synthases (PKSs) 1 are responsible for an extraordinarily large and diverse group of natural products that have important pharmaceutical applications such as antibiotic, antitumor, antifungal, anticholesterolemic and antiparasitic agents 2. PKSs are classified on the basis of their protein architecture; bacterial type I PKSs are large multifunctional polypeptides with all core enzymatic functions for elongation and modification of the carbon backbone grouped as modules. Type I PKS biosynthetic pathways are normally constructed with one module for each condensation reaction, often with additional modules that can make non-elongating modifications, or iterative modifications incorporating multiple units. The minimal functions in an elongating module are the ketosynthase (KS) domain, which acquires either a starter unit or the oligoketide from the previous module, and an acyl carrier protein (ACP) domain that holds the extender unit (most commonly malonate or methylmalonate). The KS catalyses a Claisen condensation, creating a new carbon-carbon bond in the ACP bound intermediate. Canonically, type I modules also contain an acyltransferase (AT) that loads the extender unit onto the ACP (known as cis-AT systems). However, an increasing number of known Type I PKSs, including pathways considered here, are trans-AT systems which lack integral AT domains in the extension modules but that encode one or more separate ATs to perform this function 3. After the Claisen condensation, modifications of the acyl chain may take place before transfer to the next module, most commonly β-ketoreduction (KR) alone, KR and dehydration (DH) or KR, DH and enoyl reduction (ER), yielding hydroxyl, alkene and alkane moieties respectively. In addition, trans-AT systems can introduce α-methyl groups with a methyl-transferase (MT) domain as part of the Type I module. A more complex modification found in numerous known trans-AT and several cis-type I systems occurs at the βketo group and is introduced by a transacting "HCS cassette", typically comprising an ACP, an hydroxymethylglutaryl-CoA (HMG-CoA) synthase (HCS), two proteins belonging to the crotonase superfamily and a decarboxylase. The HCS cassettes can introduce otherwise difficult modifications such as β-methyl, β-ethyl, β-methoxymethyl, cyclopropane and vinyl chloride moieties in different systems 4. This cassette normally acts on a type I module characterised by the absence of KR, DH or ER functions, often with tandemly repeated ACPs. In the myxovirescin system there are two HCS enzymes, one of which acts specifically at one of two β-branch ACPs. Understanding what signals such specific modifications is
Biochemistry, 2014
Lactimidomycin (LTM, 1) and iso-migrastatin (iso-MGS, 2) belong to the glutarimide-containing polyketide family of natural products. We previously cloned and characterized the mgs biosynthetic gene cluster from Streptomyces platensis NRRL 18993. The iso-MGS biosynthetic machinery featured an acyltransferase (AT)-less type I polyketide synthase (PKS) and three tailoring enzymes (MgsIJK). We now report cloning and characterization of the ltm biosynthetic gene cluster from Streptomyces amphibiosporus ATCC 53964, which consists of nine genes that encode an AT-less type I PKS (LtmBCDEFGHL) and one tailoring enzyme (LtmK). Inactivation of ltmE or ltmH afforded the mutant strain SB15001 or SB15002, respectively, that abolished the production of 1, as well as the three cometabolites 8,9-dihydro-LTM (14), 8,9-dihydro-8S-hydroxy-LTM (15), and 8,9-dihydro-9R-hydroxy-LTM (13). Inactivation of ltmK yielded the mutant strain SB15003 that abolished the production of 1, 13, and 15 but led to the accumulation of 14.
Catalytic self-acylation of type II polyketide synthase acyl carrier proteins
Chemistry & Biology, 1998
Background: Aromatic polyketides are synthesised in streptomycetes by the successive condensation of simple carboxylic acids, catalysed by multienzyme complexes-the polyketide synthases (PKSs). Polyketide assembly intermediates are covalently linked as thioesters to the holo-acyl carrier protein (ACP) subunit of these type II PKSs. The ACP is primed for chain elongation by the transfer of malonate from malonyl CoA. Malonylation of fatty acid synthase (FAS) ACPs is catalysed by specific malonyl transferase (MT) enzymes. The type II PKS gene clusters apparently lack genes encoding such MT proteins, however. It has been proposed that the MT subunit of the FAS in streptomycetes catalyses malonylation of both FAS and PKS ACPs in viva. Results: We demonstrate that type II PKS ACPs catalyse self-malonylation upon incubation with malonyl CoA in vitro. The self-malonylation reaction of the actinorhodin Cl 7s holo-ACP has a K, for malonyl CoA.of 219 PM and a kcat of 0.34 min-'. Complete acylation of the PKS ACPs was observed with malonyl, methylmalonyl and acetoacetyl CoAs. No reaction was observed with acetyl and butyryl CoAs and FAS ACPs did not react with any of the substrates. Recombinant FAS MT from Streptomyces coelicolor did not accelerate the rate .of malonylation.