Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization

Abstract

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 ...

Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis

“…The structure-function studies presented herein provide a picture that is likely general for C-C bond formation in polyketide chain-terminating CLCs. Combined with the recently published x-ray crystal structure of the PksA PT domain (23), these studies provide a comprehensive view of the key polyketide cyclization reactions characteristic of NR-PKSs.…”

Rational reprogramming of fungal polyketide first-ring cyclization

“…nrPKSs select different starter units by a starter unit:ACP transacylase domain (9) and mold the polyketide chains into cyclic products by regiospecific cyclizations. First-ring cyclizations are catalyzed by the product template domains (PTs) (10), whereas the polyketide chains are terminated by Claisen cyclase (11), macrolactone synthase (12) [thioesterase (TE)], or reductive release domains (2).…”

“…A crucial step during the programmed biosynthesis of aromatic polyketide natural products is the cyclization of the first ring, which is catalyzed by the PT domains of the nrPKS (10,27). This event commits the highly reactive pluripotent poly-β-ketoacyl chains to defined structural classes of the possible polyketide scaffold space.…”

“…Nevertheless, a select few fungal polyketides, including DALs like 2, feature a first-ring connectivity that is analogous to the S-type folding mode, resulting from an unorthodox C8-C3 aldol cyclization event (20). The bacterial aromatase/cylcase enzymes show little sequence similarity to fungal PT domains and feature a different protein fold and active site architecture as a prominent example of convergent evolution (10,(30)(31)(32). The sequences of fungal PT domains catalyzing F-type cyclization can be classified into seven clades according to their regiospecificity and the length of their product (29,33).…”

The International Conference of Natural Product Biosynthesis (ICNPB, 8th US–Japan seminar on the Biosynthesis of Natural Products)

“…Professor Hiroyuki Morita (Toyama University) reported the catalytic mechanism of diarylheptanoid scaffold-forming curcuminoid synthase from Oryza sativa based on its crystal structure. 7 Professor Sheryl Tsai (University of California, Irvine) presented two different mechanisms to cyclize the polyketomethylene chain by the product template domain of fungal PKS (norsolorinic acid anthrone synthase) 8 and the cyclase of type II PKS systems (tetracenomycin-producing system), respectively. 9 Dr Kenji Arakawa (Hiroshima University) described the structural elucidation and biosynthesis of the butenolide signaling molecules that induce lankacidin and lankamycin production in Streptomyces rochei.…”