Polyketide Decarboxylative Chain Termination Preceded by O-Sulfonation in Curacin A Biosynthesis

Abstract

Biosynthetic innovation in natural product systems is driven by the recruitment of new genes and enzymes into these complex pathways. Here, an unprecedented decarboxylative chain termination mechanism is described for the polyketide synthase of curacin A, an anticancer lead compound isolated from the marine cyanobacterium Lyngbya majuscula. The unusual chain termination module containing adjacent sulfotransferase (ST) and thioesterase (TE) catalytic domains embedded in CurM was biochemically characterized. The TE was proved to catalyze a hydrolytic chain release of the polyketide chain elongation intermediate. Moreover, a selective ST-mediated sulfonation of the (R)-β-hydroxyl group was found to precede TE-mediated hydrolysis, triggering a successive decarboxylative elimination and resulting in the formation of a rare terminal olefin in the final metabolite.Curacin A ( Figure 1A), a marine cyanobacterial metabolite isolated from marine cyanobacterium Lyngbya majuscula, is a mixed-polyketide nonribosomal-peptide natural product with potent anticancer activities. 1 The biosynthetic pathway generates a series of intermediates with increasing hydrophobicity leading to the final product. STs catalyze transfer of a sulfonate group from the donor, adenosine 3′-phosphate 5′-phosphosulfate (PAPS), to a hydroxyl or amine group on receptors ranging from proteins to small molecules.11 They are involved in a broad range of biological processes, such as detoxification, hormone regulation, drug metabolism, signaling pathways, and others.11 Typically, a polyketide synthase or non-ribosomal peptide synthetase TE catalyzes a chain termination process by releasing a full-length acyl, peptidyl, or hybrid chain from the carrier protein phosphopantetheine thiol group as a free acid, or employs an intramolecular nucleophile (e.g., hydroxyl or amine group) to generate a macrolactone or macrolactam product.12 -14 Based on the predicted full-length intermediate tethered to the CurM ACP ( Figure 1A), a series of reactions including thioester hydrolysis, decarboxylation and dehydration are required to form the terminal olefin moiety. According to the canonical functions of ST and TE, the CurM ST was predicted to transfer a sulfonate group to the β-hydroxyl of the full-length intermediate, and the CurM TE was presumed to release the intermediate from the ACP as a free acid. However, how these enzymes are coordinated to render the terminal olefin formation has remained elusive. Thus, we were motivated to pursue biochemical studies to elucidate the mechanisms of this novel chain termination process.Our bioinformatics analysis and protein solubility tests suggested that the previously sequenced curM TE region 2 might have been partially replaced with exogenous DNA during construction of the genomic DNA library of L. majuscula. Thus, we sequenced curM and additional parts of the 3' flanking region (GenBank accession no. GQ412749) of the cur cluster in another cosmid (pLM14), from the L. majuscula genomic DNA library 15 and compared the ...

Terminal Alkene Formation by the Thioesterase of Curacin A Biosynthesis

“…5B). The TE wild type and variants were tested using a one-pot, multistep assay (9) in which recombinant CurM ACP was loaded with a synthetic substrate mimic (Fig. 1B) and reacted sequentially with recombinant CurM ST and CurM TE.…”

“…All constructs were verified by sequencing. The CurM ACP and ST expression plasmids were previously described (9).…”

“…Enzyme Assay-CurM TE activity was assayed using a modification of our previous protocol (9). Apo-ACP was loaded with a substrate analog by a 2-h incubation of 50 M apo-ACP, 100 M (3R)-hydroxy-5-methoxytetradecanoyl-CoA (9), 10 M S. verticillus Svp (36), 10 mM MgCl 2 , 100 mM Tris, pH 7.9, at 30°C.…”

“…The hybrid PKS/NRPS assembly line pathway for curacin A (4) generates several unusual chemical groups in addition to the terminal alkene, including a cyclopropyl ring, a thiazoline ring, and a cis double bond. We have investigated the biosynthetic steps leading to several of these segments (5)(6)(7)(8)(9). Herein we investigate the structural basis for the unique offloading strategy leading to the terminal alkene in the curacin A molecule.…”

“…Using a simplified analog of the penultimate pathway intermediate, we recently demonstrated that off-loading and terminal alkene formation require ST-mediated sulfonation of the ␤-hydroxyl group from the PAPS cofactor (9) (Fig. 1A).…”