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

Abstract

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.

Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases

“…The last step of BDL scaffold biosynthesis is the release of the product, catalyzed by an O-C bond-forming thioesterase (TE) domain of the nrPKS. TE domains form the BDL macrolactone using the ω-1 alcohol as a nucleophile, but may use alternative nucleophiles such as the C9 enol to yield α-pyrones or water or alcohols from the media to form acyl resorcylic acids (ARAs), acyl dihydroxyphenylacetic acids (ADAs), and their esters (18)(19)(20)(21)(22). iPKSs that produce BDLs in the RAL 14 , RAL 12 , and DAL 12 subclasses have been characterized and reconstituted both in vivo by heterologous expression in yeast and in vitro using isolated recombinant iPKS enzymes (11)(12)(13)(14)(23)(24)(25).…”

Characterization of a fungal thioesterase having claisen cyclase and deacetylase activities in melanin biosynthesis

“…A homology model of Pks1 TE was prepared using the recently determined 1.7 Å crystal structure of the PksA TE domain (PDB 3ILS) as the template, which shares 37% sequence identity and 53% sequence similarity with the Pks1 TE ( Figure 5B) (Korman, et al, 2010). In comparison to Pks1, the PksA TE accepts a bicyclic C 20 substrate with a hexyl starterunit side chain and catalyzes an analogous Claisen cyclization to close the third ring of norsolorinic acid anthrone .…”

“…The Claisen/Dieckmann cyclase class of TE domains (TE/CLC), which catalyzes C-C bond formation in contrast to the classical hydrolytic release of fatty acids, was originally identified in the wA naphthopyrone synthase from A. nidulans . Further insight into this class of TEs was advanced through determination of the crystal structure of a dissected TE/CLC monodomain from the norsolorinic acid anthrone synthase, PksA, from A. parasiticus (Korman, et al, 2010). Inactivating mutation or deletion of TE/CLC domains generally results in spontaneous O-C cyclization to release full-length pyrone shunt products .…”

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).…”