Biosynthetic Pathway and Gene Cluster Analysis of Curacin A, an Antitubulin Natural Product from the Tropical Marine Cyanobacterium Lyngbya majuscula

Abstract

Curacin A (1) is a potent cancer cell toxin obtained from strains of the tropical marine cyanobacterium Lyngbya majuscula found in Curaçao. Its structure is unique in that it contains the sequential positioning of a thiazoline and cyclopropyl ring, and it exerts its potent cell toxicity through interaction with the colchicine drug binding site on microtubules. A series of stable isotope-labeled precursors were fed to cultures of curacin A-producing strains and, following NMR analysis, allowed determination of the metabolic origin of all atoms in the natural product (one cysteine, 10 acetate units, two S-adenosyl methionine-derived methyl groups) as well as several unique mechanistic insights. Moreover, these incorporation experiments facilitated an effective gene cloning strategy that allowed identification and sequencing of the approximately 64 kb putative curacin A gene cluster. The metabolic system is comprised of a nonribosomal peptide synthetase (NRPS) and multiple polyketide synthases (PKSs) and shows a very high level of collinearity between genes in the cluster and the predicted biochemical steps required for curacin biosynthesis. Unique features of the cluster include (1) all but one of the PKSs are monomodular multifunctional proteins, (2) a unique gene cassette that contains an HMG-CoA synthase likely responsible for formation of the cyclopropyl ring, and (3) a terminating motif that is predicted to function in both product release and terminal dehydrative decarboxylation.

Convergence of isoprene and polyketide biosynthetic machinery: Isoprenyl- S -carrier proteins in the pksX pathway of Bacillus subtilis

“…Consistent with this proposed series of events, when we treated [2-14 C]Mal-S-AcpK and Acac-S-PksL-T2 with PksF, PksG, PksH, and PksI, we observed stable radiolabeling of PksL-T2. In contrast, when the same experiment was performed with [1,[3][4][5][6][7][8][9][10][11][12][13][14] C]Mal-SAcpK, we observed significant loss of radiolabel from PksL-T2 when PksH and PksI were added ( Fig. 5 and Fig.…”

“…No decarboxylation was detected when Mal-S-PksL-T2 was incubated with PksF, demonstrating that it is selective for Mal-S-AcpK (data not shown). Treatment of [1,[3][4][5][6][7][8][9][10][11][12][13][14] C]Mal-S-AcpK with PksF gave similar results, with rapid PksF-dependent loss of radiolabel observed, to form [1-14 C]Ac-S-AcpK at a rate of 5.5 M⅐min Ϫ1 (Figs. 8 and 9, which are published as supporting information on the PNAS web site).…”

“…We also identify roles for the freestanding carrier protein AcpK and the tandem T-T didomain in PksL. Collections of proteins similar to AcpK, PksFGHI, and tandem carrier domains such as those found in PksL have also been found in the curacin, jamaicamide, mupriocin, antibiotic TA, and pederin biosynthetic clusters (10)(11)(12)(13)(14)(15)(16)(17)(18)(19), and they appear to encode the biosyntheses of natural products that universally contain a single-carbon ␤-branch (18).…”

“…Although the pksX cluster has been proposed to encode the biosynthesis of difficidin (7,9,10), recent work has demonstrated that the NRPS portion of the gene cluster is active, disqualifying difficidin, which does not contain any amines, as a candidate for the biosynthetic product of this cluster (11). Long thought to be cryptic, there is now evidence that it is responsible for the biosynthesis of a product that kills streptomycetes (P. D. Straight, M. A. Fischbach, C.T.W., and R. Kolter, unpublished results).…”

The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis

“…We then used an NMR overlay technique to identify compounds present in extracts from strains with the pksX gene cluster (pksX ϩ ) and absent in extracts from strains in which the cluster was deleted (pksX Ϫ ) (7). The structure of bacillaene is remarkable given that it is linear, has a high degree of conjugation with several cis double bonds, and incorporates a ␤-branch, a feature seen in only a few other polyketides (8)(9)(10)(11)(12). Based on bacillaene's structure, we were able to annotate further the pksX gene cluster, which contains two unusual split modules-modules in which the enzymatic domains of a single polyketide module are split between two separate proteins.…”