Terminal alkene formation by the thioesterase of curacin A biosynthesis: structure of a decarboxylating thioesterase.

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 ß-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., Håkansson, K., Smith, J. L., and Sherman, D. H. (2009) J. Am. Chem. Soc. 131, 16033-16035). With low sequence identity to other PKS TEs (<15%), the curacin TE represents a new thioesterase subfamily. The 1.7-Å curacin TE crystal structure reveals how the familiar α/ß-hydrolase architecture is adapted to specificity for ß-sulfated substrates. A Ser-His-Glu catalytic triad is centered in an open active site cleft between the core domain and a lid subdomain. Unlike TEs from other PKSs, the lid is fixed in an open conformation on one side by dimer contacts of a protruding helix and on the other side by an arginine anchor from the lid into the core. Adjacent to the catalytic triad, another arginine residue is positioned to recognize the substrate ß-sulfate group. The essential features of the curacin TE are conserved in sequences of five other putative bacterial ACP-ST-TE tridomains. Formation of a sulfate leaving group as a biosynthetic strategy to facilitate acyl chain decarboxylation is of potential value as a route to hydrocarbon biofuels.

Insights from the sea: structural biology of marine polyketide synthases.

The world's oceans are a rich source of natural products with extremely interesting chemistry. Biosynthetic pathways have been worked out for a few, and the story is being enriched with crystal structures of interesting pathway enzymes. By far, the greatest number of structural insights from marine biosynthetic pathways has originated with studies of curacin A, a poster child for interesting marine chemistry with its cyclopropane and thiazoline rings, internal cis double bond, and terminal alkene. Using the curacin A pathway as a model, structural details are now available for a novel loading enzyme with remarkable dual decarboxylase and acetyltransferase activities, an Fe(2+)/α-ketoglutarate-dependent halogenase that dictates substrate binding order through conformational changes, a decarboxylase that establishes regiochemistry for cyclopropane formation, and a thioesterase with specificity for ß-sulfated substrates that lead to terminal alkene offloading. The four curacin A pathway dehydratases reveal an intrinsic flexibility that may accommodate bulky or stiff polyketide intermediates. In the salinosporamide A pathway, active site volume determines the halide specificity of a halogenase that catalyzes for the synthesis of a halogenated building block. Structures of a number of putative polyketide cyclases may help in understanding reaction mechanisms and substrate specificities although their substrates are presently unknown.

Polyketide decarboxylative chain termination preceded by o-sulfonation in curacin a biosynthesis.

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)-beta-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.

Structure and activity of DmmA, a marine haloalkane dehalogenase.

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) = 0.24 ± 0.05 mM, k(cat) = 2.4 ± 0.1 s(-1) ). 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.