Poly Specific trans -Acyltransferase Machinery Revealed via Engineered Acyl-CoA Synthetases (original) (raw)
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Nature communications, 2016
Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular ba...
Reprogramming Acyl Carrier Protein Interactions of an Acyl-CoA Promiscuous trans-Acyltransferase
Chemistry & Biology, 2014
Protein interactions between acyl carrier proteins (ACPs) and trans-acting acyltransferase domains (trans-ATs) are critical for regioselective extender unit installation by many polyketide synthases, yet little is known regarding the specificity of these interactions, particularly for trans-ATs with unusual extender unit specificities. Currently, the best-studied trans-AT with nonmalonyl specificity is KirCII from kirromycin biosynthesis. Here, we developed an assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a noncognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions and for engineering modular polyketide synthases to produce analogs.
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...
The Journal of biological chemistry, 2015
Understanding the biosynthetic mechanism of the atypical polyketide extender unit is important to develop bioactive natural products. Reveromycin (RM)-derivatives produced by Streptomyces sp. SN-593 possess several aliphatic extender units. Here, we studied the molecular basis of 2-alkylmalonyl-CoA formation by analyzing the revR and revS genes, which form a transcriptional unit with the revT gene, a crotonyl-CoA reductase/carboxylase homolog. We mainly focused on uncharacterized adenylate-forming enzyme (RevS). revS gene disruption resulted in the reduction of all RM-derivatives, while reintroduction of the gene restored the yield of RMs. Although RevS was classified in the fatty acyl-AMP ligase (FAAL) clade based on phylogenetic analysis, biochemical characterization revealed that the enzyme catalyzed middle chain fatty acyl-CoA ligase (FACL) but not FAAL activity, suggesting the molecular evolution for acyl-CoA biosynthesis. Moreover, we examined the in vitro conversion of fatty ...
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
2019
ABSTRACTAcyltransferase (AT)-less type I polyketide synthases (PKSs) produce complex natural products due to the presence of many unique tailoring enzymes. The 3-hydroxy-3-methylglutaryl coenzyme A synthases (HCSs) are responsible for β-alkylation of the growing polyketide intermediates in AT-less type I PKSs. In this study, we discovered a large group of HCSs, closely associated with the characterized and orphan AT-less type I PKSs throughin silicogenome mining, sequence and genome neighborhood network analysis. Using HCS-based probes, the survey of 1207 inhouse strains and 18 soil samples from different geological locations revealed the vast diversity of HCS-containing AT-less type I PKSs. The presence of HCSs in many AT-less type I PKSs suggests their co-evolutionary relationship. Our study should inspire future efforts to discover new polyketide natural products from AT-less type I PKSs.
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