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 ...

Curacin A is a polyketide synthase (PKS)-non-ribosomal peptide synthetase-derived natural product with potent anticancer properties generated by the marine cyanobacterium Lyngbya majuscula. Type I modular PKS assembly lines typically employ a thioesterase (TE) domain to off-load carboxylic acid or macrolactone products from an adjacent acyl carrier protein (ACP) domain. In a striking departure from this scheme the curacin A PKS employs tandem sulfotransferase and TE domains to form a terminal alkene moiety. Sulfotransferase sulfonation of ␤-hydroxy-acyl-ACP is followed by TE hydrolysis, decarboxylation, and sulfate elimination (Gu, L., Wang, B., Kulkarni, A., Gehret, J. J., Lloyd, K. R., Gerwick, L., Gerwick, W. H., Wipf, P.

DmmA is a haloalkane dehalogenase (HLD) identified and characterized from the metagenomic DNA of a marine microbial consortium. Dehalogenase activity was detected with 1,3-dibromopropane as substrate, with steady-state kinetic parameters typical of HLDs (K m 5 0.24 6 0.05 mM, k cat 5 2.4 6 0.1 s 21). The 2.2-Å crystal structure of DmmA revealed a fold and active site similar to other HLDs, but with a substantially larger active site binding pocket, suggestive of an ability to act on bulky substrates. This enhanced cavity was shown to accept a range of linear and cyclic substrates, suggesting that DmmA will contribute to the expanding industrial applications of HLDs.