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

Abstract

The pksX gene cluster from Bacillus subtilis is predicted to encode the biosynthesis of an as yet uncharacterized hybrid nonribosomal peptide͞polyketide secondary metabolite. We used a combination of biochemical and mass spectrometric techniques to assign functional roles to the proteins AcpK, PksC, PksL, PksF, PksG, PksH, and PksI, and we conclude that they act to incorporate an acetate-derived ␤-methyl branch on an acetoacetyl-S-carrier protein and ultimately generate a ⌬ 2 -isoprenyl-S-carrier protein. This work highlights the power of mass spectrometry to elucidate the functions of orphan biosynthetic enzymes, and it details a mechanism by which single-carbon ␤-branches can be inserted into polyketide-like structures. This pathway represents a noncanonical route to the construction of prenyl units and serves as a prototype for the intersection of isoprenoid and polyketide biosynthetic manifolds in other natural product biosynthetic pathways.mass spectrometry ͉ orphan gene cluster ͉ hybrid nonribosomal peptide͞polyketide ͉ polyketide methylation P olyketides and nonribosomal peptides are classes of secondary metabolites that are synthesized by the iterative coupling of malonyl (Mal) derivatives and amino acids, respectively. The catalytic machinery responsible for the biosynthesis of these natural products comprises modular synthases that incorporate, and often subsequently tailor, monomer units into the growing small molecule by a thiotemplated mechanism. Often, the sequence of protein domains and modules is colinear with respect to the sequence of biosynthetic reactions catalyzed by the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) machinery (1-3). Because of their highly modular nature, the small-molecule products of PKSs, NRPSs, and hybrid NRPS-PKS systems can often be predicted by bioinformatic approaches, and there have been recent experimental validations of such predictions (4-6). However, for many cases in which the biosynthetic product encoded by a given gene cluster is unknown, functional characterization of the synthase by bioinformatic methods alone is difficult or impossible.One such orphan cluster for which the product structure is unknown is the hybrid NRPS-PKS pksX cluster from Bacillus subtilis (7-9). 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). Additionally, this cluster does not follow the usual NRPS-PKS colinearity rules because it has only three trans-acting acyltransferases that may load up to 17 of the 20 predicted thiolation domains of the cluster.We were intrigued by a series of predicted ORFs withi...

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“…This recently described type of PKS protein does not contain an AT domain in each module, as in most cases, but instead ATs on a separate protein load malonate units onto acyl-carrier proteins (ACPs) on the PKS (21). Further accessory proteins are present that would be responsible for β-methylation at position 5 (PtzHIJG), in analogy to the bacillaene pathway (40), and for in trans enoyl reduction (PtzQ), as found in the corallopyronin pathway (40,41), among others (SI Appendix, Table S2). Despite being distributed in separate loci, the ptz genes clearly belong to the same biosynthetic pathway.…”

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

“…The resulting methyl branch lies at a ␤-position in the middle of the hexaene moiety. In contrast to the more common strategy of ␣-methylation by S-adenosyl methionine-dependent methyltransferase domains (such as those in PksM and PksR), ␤-alkylation requires the action of five enzymes and one external T domain (8,19).…”

“…The bacillaene synthase is unique even among trans-AT synthases because it encodes three free-standing AT domains (PksCDE), one of which is fused to a predicted flavin mononucleotide-dependent oxidoreductase (PksE). PksC has been characterized as a malonyl-CoA-specific AT that accepts AcpK as a thiolation (T) domain substrate (8), but it is unknown whether PksC services other T domains in the PksX synthase, and PksD and PksE have not yet been characterized.…”

“…2C) of proteins that use a variation of standard isoprene biosynthesis to construct a ⌬ 2 -isoprenyl-S-T branch on the T 2 motif of PksL from a ␤-ketoacyl-S-T intermediate (8). The resulting methyl branch lies at a ␤-position in the middle of the hexaene moiety.…”

“…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.…”