Polyketides are a class of natural products with diverse structures and biological activities. The structural variability of aromatic products of fungal nonreducing, multidomain iterative polyketide synthases (NR-PKS group of IPKSs) results from regiospecific cyclizations of reactive poly-β-keto intermediates [1][2][3] . How poly-β-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently 4 . A product template (PT) domain is responsible for controlling specific aldol cyclization and aromatization of these mature polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 Å crystal structure and mutational studies of a dissected PT monodomain from PksA, the NR-PKS that initiates the biosynthesis of the potent hepatocarcinogen aflatoxin B 1 in Aspergillus parasiticus. Despite having minimal sequence similarity to known enzymes, the structure displays a distinct 'double hot dog' (DHD) fold. Co-crystal structures with palmitate or a bicyclic substrate mimic illustrate that PT can bind both linear and bicyclic polyketides. Docking and mutagenesis studies reveal residues important for substrate binding and catalysis, and identify a phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber. Sequence similarity and extensive conservation of active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS cyclization specificity.Aflatoxin B 1 (Fig. 1a, 3) biosynthesis is initiated by the NR-PKS PksA, which accepts a hexanoyl starter unit from a dedicated fungal fatty acid synthase (FAS) and extends it through seven iterative, malonyl-derived ketide extensions to norsolorinic acid anthrone ((1), noranthrone) (Fig. 1a)5. Application of the Udwary-Merski algorithm (UMA) afforded the unanticipated insight that PksA was composed of two unrecognized domains, in additionCorrespondence and requests for materials should be addressed to C.A.T. (ctownsend@jhu.edu) or S.-C.T. (sctsai@uci.edu). * These authors contributed equally to this work.Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. (Fig. 1a, c) 4 . When the TE domain was absent, naphthopyrone (4) was produced by self-condensation of the stalled bicyclic PT product 7 (Fig. 1b). Author ContributionsWe crystallized PT in P2 1 2 1 2 1 and P4 1 2 1 2 space groups (Supplementary Table 1) and solved the PT crystal structure by multi-wavelength anomalous dispersion (MAD) using selenomethionine-derivatized PT followed by phase extension with the native data to 1.8 Å.The crystal structures from both crystal forms showed distinct ...

Polyketides are a class of natural products with highly diverse chemical structures and pharmaceutical activities. Polyketide cyclization, promoted by the aromatase/cyclase (ARO/CYC), helps diversify aromatic polyketides. How the ARO/CYC promotes highly specific cyclization is not well understood because of the lack of a first-ring ARO/CYC structure. The 1.9 Å crystal structure of Tcm ARO/CYC reveals that the enzyme belongs to the Bet v1-like superfamily (or STAR domain family) with a helix–grip fold, and contains a highly conserved interior pocket. Docking, mutagenesis, and an in vivo assay show that the size, shape, and composition of the pocket are important to orient and specifically fold the polyketide chain for C9-C14 first-ring and C7-C16 second-ring cyclizations. Two pocket residues, R69 and Y35, were found to be essential for promoting first- and second-ring cyclization specificity. Different pocket residue mutations affected the polyketide product distribution. A mechanism is proposed based on the structure-mutation-docking results. These results strongly suggest that the regiospecific cyclizations of the first two rings and subsequent aromatizations take place in the interior pocket. The chemical insights gleaned from this work pave the foundation toward defining the molecular rules for the ARO/CYC cyclization specificity, whose rational control will be important for future endeavors in the engineered biosynthesis of novel anticancer and antibiotic aromatic polyketides.

Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. Here we design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the production of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addition of glucose. We obtain conversion yields >95% and titres >15 g l−1. The titres are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals.

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).* Author to whom correspondence should be addressed. Phone 949-824-4486, e-mail sctsai@uci.edu, fax 949-824-8552. ⊥ Department of Molecular Biology and Biochemistry. § Department of Chemistry.1 Abbreviations: KR, ketoreductase; FabG, β-ketoacyl [acyl carrier protein] reductase; Act, actinorhodin; PKS, polyketide synthase; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; SDR, short-chain dehydrogenase/reductase; T3HNR, 1,3,8-trihydro...

Polyketide natural products possess diverse architectures and biological functions and share a subset of biosynthetic steps with fatty acid synthesis. The final transformation catalyzed by both polyketide synthases (PKSs) and fatty acid synthases is most often carried out by a thioesterase (TE). The synthetic versatility of TE domains in fungal nonreducing, iterative PKSs (NR-PKSs) has been shown to extend to Claisen cyclase (CLC) chemistry by catalyzing C–C ring closure reactions as opposed to thioester hydrolysis or O–C/N–C macrocyclization observed in previously reported TE structures. Catalysis of C–C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, but how this activity was acquired has remained a mystery. We report the biochemical and structural analyses of the TE/CLC domain in polyketide synthase A, the multidomain PKS central to the biosynthesis of aflatoxin B 1 , a potent environmental carcinogen. Mutagenesis experiments confirm the predicted identity of the catalytic triad and its role in catalyzing the final Claisen-type cyclization to the aflatoxin precursor, norsolorinic acid anthrone. The 1.7 Å crystal structure displays an α/β-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber. We propose that a key rotation of the substrate side chain coupled to a protein conformational change from the open to closed form spatially governs substrate positioning and C–C cyclization. The biochemical studies, the 1.7 Å crystal structure of the TE/CLC domain, and intermediate modeling afford the first mechanistic insights into this widely distributed C–C bond-forming class of TEs.