Structural and Biochemical Analyses of Regio- and Stereospecificities Observed in a Type II Polyketide Ketoreductase (original) (raw)
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The Determinants of Activity and Specificity in Actinorhodin Type II Polyketide Ketoreductase
Chemistry & Biology, 2013
Bacterial aromatic polyketides include many therapeutic agents and are biosynthesized by type II polyketide synthases (PKSs). In the actinorhodin type II PKS, the first polyketide modification is a regiospecific C9-carbonyl reduction, catalyzed by the ketoreductase (actKR). Our previous studies identified the actKR 94-PGG-96 motif as a determinant of stereospecificity (Javidpour, et al., 2011). The molecular basis for reduction regiospecificity is, however, not well understood. In this study, we examined the activities of 20 actKR mutants through a combination of kinetic studies, PKS reconstitution, and structural analyses. Residues have been identified which are necessary for substrate interaction, and these observations have suggested a structural model for this reaction. Polyketides dock at the KR surface and are steered into the enzyme pocket where C7-C12 cyclization is mediated by the KR before C9-ketoreduction can occur. These molecular features can potentially serve as engineering targets for the biosynthesis of novel, reduced polyketides. HIGHLIGHTS • ActKR surface arginines are important for ACP-binding and activity toward polyketides • In contrast to the S-specific P94L actKR, mutant V151L displays R-stereospecificity • ActKR is proposed to mediate C7-C12 polyketide cyclization prior to C9-ketoreduction
Inhibition Kinetics and Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase
Biochemistry, 2008
Type II polyketides are a class of natural products that include pharmaceutically important aromatic compounds such as the antibiotic tetracycline and antitumor compound doxorubicin. The type II polyketide synthase (PKS) is a complex consisting of 5-10 standalone domains homologous to fatty acid synthase (FAS). Polyketide ketoreductase (KR) provides regio-and stereochemical diversity during the reduction. How the type II polyketide KR specifically reduces only the C9 carbonyl group is not well understood. The cocrystal structures of actinorhodin polyketide ketoreductase (actKR) bound with NADPH or NADP + and the inhibitor emodin were solved with the wild type and P94L mutant of actKR, revealing the first observation of a bent p-quinone in an enzyme active site. Molecular dynamics simulation help explain the origin of the bent geometry. Extensive screening for in vitro substrates shows that unlike FAS KR, the actKR prefers bicyclic substrates. Inhibition kinetics indicate that actKR follows an ordered Bi Bi mechanism. Together with docking simulations that identified a potential phosphopantetheine binding groove, the structural and functional studies reveal that the C9 specificity is a result of active site geometry and substrate ring constraints. The results lay the foundation for the design of novel aromatic polyketide natural products with different reduction patterns. The pharmaceutical potential of bacterial or fungal natural products is illustrated by the large number of compounds that are clinically applied as therapeutics. Many pharmaceutically relevant natural products are derived from polyketides and are used as antibiotic (tetracyclines, actinorhodin), anticancer (doxorubicin), antiviral (rebeccamycin derivatives), and cholesterollowering (statins) compounds (1). The antibiotics such as tetracycline and actinorhodin are biosynthesized from acyl-CoA thiosters by type II polyketide synthases (PKSs 1), which are structurally and functionally related to the type II fatty acid synthase (FAS) (2). Compared to the type I FAS and PKS, which have enzyme domains covalently linked together, the type II FAS and PKS consist of 5-10 standalone enzymes that catalyze the condensation of malonyl extender units iteratively, followed by chain modifications, to produce the aromatic polyketides (3,4). † This work is supported by the Pew Foundation and National Institute of General Medicinal Sciences (NIGMS R01GM076330). ‡ The atomic coordinates have been deposited in the Protein Data Bank (accession code 2RH4, 2RHC, and 2RHR).
The Crystal Structure of the actIII Actinorhodin Polyketide Reductase
Structure, 2004
is the ketosynthase (KS), chain length factor (CLF), and the acyl carrier protein (ACP) (McDaniel et al., 1994, University Walk Bristol, BS8 1TD 1995). The KS catalyzes the decarboxylative condensation of malonyl extender units, while CLF is involved in United Kingdom 2 School of Chemistry formation of acetyl ACP (from the decarboxylation of malonyl-ACP) (Bisang et al., 1999) and may also influ-University of Bristol Cantock's Close ence the chain length of the final polyketide. The growing polyketide is attached as a thioester via a phosphopan-Bristol BS8 1TS United Kingdom tetheine group to a serine at the N terminus of helix 2 in ACP. The ACP may have several roles: to sequester the polyketide chain, protecting it from acid/base enolization and cyclization, and to act as an extended sub-Summary strate for the other enzymes in the polyketide synthase. The molecular details of the interactions between ACP We have determined the 2.5 Å crystal structure of an active, tetrameric Streptomyces coelicolor type II poly-and the type II enzymes are only just beginning to be understood (Worsham et al., 2003; Zhang et al., 2001). ketide ketoreductase (actIII) with its bound cofactor, NADP ؉. This structure shows a Rossman dinucleotide It is evident, however, that in any one biosynthetic pathway, the ACP has the task of recognizing many different binding fold characteristic of SDR enzymes. Of two subunits in the crystallographic asymmetric unit, one enzymes. The type II aromatic PKS from Streptomyces coelico-is closed around the active site. Formate is observed in the open subunit, indicating possible carbonyl binding lor that produces actinorhodin (act) has been extensively studied (Hopwood, 1997). As well as producing a sites of the polyketide intermediate. Unlike previous models we observe crystal contacts that may mimic 16 carbon polyketide chain, the minimal system can partially control the first cyclization, though products the KR-ACP interactions that may drive active site opening. Based on these observations, we have con-from aberrant cyclizations may also be observed (Fu et al., 1994a, 1994b). The act minimal PKS produces two structed a model for ACP and polyketide binding. We propose that binding of ACP triggers a conformational products, SEK4 (Figure 1) with the correct first cyclization (C7-C12), and SEK4b with an alternate C10-C15 change from the closed to the open, active form of the enzyme. The polyketide chain enters the active ring closure (Fu et al., 1994b). The act ketoreductase (act KR) is the first enzyme to modify the polyketide, its site and reduction occurs. The model also suggests a general mechanism for ACP recognition which is primary function being to reduce the carbonyl group at C-9. Addition of the act KR to the minimal system, both applicable to a range of protein families. in vivo and in vitro, yields predominantly mutactin, which arises from the correctly cyclized and reduced interme-Introduction diate (Figure 1) (Fu et al., 1994c; Zhang et al., 1990). The elimination of incorrectly cyclized products suggests Polyketides are a chemically diverse group of secondary metabolites produced by bacteria, fungi, plants, and that the KR may aid the minimal complex in directing the correct aldol condensation between C7 and C12. marine organisms (Hopwood, 1990, 1997; Staunton and Weissman, 2001). These natural products are of enor-The mechanisms for regiospecific control of cyclization and reduction are unknown at present. In the presence mous pharmaceutical interest and include antibiotic of the act KR, there is an overall increase in polyketide (oxytetracycline, a tetracycline), anticancer (doxorubiproduction when compared with the minimal system, cin, an anthracycline), and antifungal (resveratrol, a stilpossibly suggesting stabilization of the complex or more bene) compounds. Analogous to fatty acid synthases efficient intermediate channelling (Zawada and Khosla, (FASs) (Smith, 1994), the polyketide synthase (PKS) bio-1999). The influence of the KR in other minimal systems synthetic complex is organized as either a covalent (type has also been reported. An increase in misfolded polyke-I) or noncovalent (type II) assembly of catalytic domains. tides is seen in ketoreductase-deficient mutants of some Both fatty acid and polyketide biosynthesis proceed via synthases (Kunnari et al., 1999), and in extreme cases the iterative decarboxylative condensation of (normally) the minimal system produces no polyketide in the abmalonyl or methyl-malonyl extender units to a starter sence of the KR (Hertweck et al., 2004). unit, usually acetate. Polyketide synthases achieve their To date, the structures of five type II aromatic PKS structural diversity by selecting primer and extension components have been solved. These are the X-ray structures of the unusual priming -ketosynthase
Biochemistry
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 A 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 alpha6-alpha7 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...
Mechanistic insights into the biosynthesis of polyketide antibiotics
2006
Anthracyclines are a group of aromatic polyketide compounds with significant medical importance due to their antineoplastic properties. Doxorubicin and daunorubicin, members of this family are among the two most commonly used anticancer drugs. These compounds exhibit severe side effects like cardiotoxicity and multi-drug resistance. A promising approach towards the production of modified anthracyclines with improved toxicity profiles appears to be combinatorial biosynthesis, including the redesign of biosynthetic enzymes; however, structural and mechanistic information of the biosynthetic enzymes is necessary for the redesigning approach. 2.2.3 The overall fold of AknOx 2.2.4 AknOx belongs to PCMH superfamily 2.2.4.1 The conserved F-domain and FAD binding features in PCMH superfamily 2.2.4.2 Diversity in the structure of the substrate binding domain is observed in PCMH superfamily 2.2.5 FAD binding site in AknOx 2.2.6 Covalent flavinylation in AknOx and other flavoenzymes 2.2.6.1 Categories of covalent flavinylation observed in different flavoenzymes 2.2.6.2 The flavinylation observed in AknOx and PCMH superfamily 2.2.7 Ligand binding features and active site 2.2.8 Catalytic mechanism of AknOx 2.2.9 Comparison of catalytic properties in PCMH superfamily 3 Conclusions 3.1 SnoaL and AknH 3.2 AknOx References Acknowledgements
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.
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.
The Stereochemistry of Complex Polyketide Biosynthesis by Modular Polyketide Synthases
Molecules, 2011
Polyketides are a diverse class of medically important natural products whose biosynthesis is catalysed by polyketide synthases (PKSs), in a fashion highly analogous to fatty acid biosynthesis. In modular PKSs, the polyketide chain is assembled by the successive condensation of activated carboxylic acid-derived units, where chain extension occurs with the intermediates remaining covalently bound to the enzyme, with the growing polyketide tethered to an acyl carrier domain (ACP). Carboxylated acyl-CoA precursors serve as activated donors that are selected by the acyltransferase domain (AT) providing extender units that are added to the growing chain by condensation catalysed by the ketosynthase domain (KS). The action of ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) activities can result in unreduced, partially reduced, or fully reduced centres within the polyketide chain depending on which of these enzymes are present and active. The PKS-catalysed assembly process generates stereochemical diversity, because carbon-carbon double bonds may have either cis-or trans-geometry, and because of the chirality of centres bearing hydroxyl groups (where they are retained) and branching methyl groups (the latter arising from use of propionate extender units). This review shall