PksA, which initiates biosynthesis of the environmental carcinogen aflatoxin B1, is one of the multidomain iterative polyketide synthases (IPKSs), a large, poorly understood family of biosynthetic enzymes. We found that dissection of PksA and its reconstitution from selected sets of domains allows the accumulation and characterization of advanced octaketide intermediates bound to the enzyme, permitting the reactions controlled by individual catalytic domains to be identified. A product template (PT) domain unites with the ketosynthase and thioesterase in this IPKS system to assemble precisely seven malonyl-derived building blocks to a hexanoyl starter unit and mediate a specific cyclization cascade. Because the PT domain is common among nonreducing IPKSs, these mechanistic features should prove to be general for IPKS-catalyzed production of aromatic polyketides.Tens of thousands of natural products are known from microorganisms, plants, and animals that provide hormones, toxins, flavors and fragrances, pigments, drugs, and other materials of commercial value. A handful of biosynthetic pathways give rise to this rich diversity of useful structures. Among these, polyketides are synthesized from simple acyl-coenzyme A (acylCoA) substrates by polyketide synthases (PKSs) (1). We understand a great deal about the function of giant modular PKSs that synthesize complex products, for example, the antibiotic erythromycin and the immunosup-pressant rapamycin (2). Each catalytic domain in these multidomain (type I) megaproteins is used once in an overall "assembly-line" process as a growing intermediate is advanced along the enzyme to yield a product. In contrast to these bacterial systems, in eukaryotes iterative PKSs (IPKSs) are generally the rule where a markedly smaller number of catalytic domains are similarly fused but individually reused in multiple catalytic cycles (iteration) that are "programmed" to yield specific products. How programming is achieved is a central unanswered question of iterative catalysis.We undertook a "deconstruction" approach by domain dissection and reassembly of PksA, the nonreducing IPKS of aflatoxin biosynthesis, to reveal the global division of labor among the domains in these macromolecular machines to control polyketide chain length, cyclization of an extended poly-β-keto intermediate, and product release. A domain hypothesized to be a "product template" (PT) has been discovered to play a central role in product formation. These studies are combined with high-resolution mass spectrometry (MS) to monitor the highly †To whom correspondence should be addressed.

The neoclerodane diterpene salvinorin A (1) was isolated in 1982 from the rare mint SalVia diVinorum, indigenous to Oaxaca, Mexico. 1 Recent efforts established salvinorin A as a potent and selective κ opioid receptor agonist, the only non-alkaloid psychoactive substance, and the most potent naturally occurring hallucinogen. 2 As a result of its therapeutic potential, renewed isolation efforts have discovered a number of related salvinorin congeners, 3 and a number of analogues of 1 have been prepared by semisynthesis to probe the pharmacophore and mode of binding. 4 This communication describes the first synthesis of this natural product.Construction of the tricyclic salvinorin core is predicated on the proposed transannular 5 Michael reaction cascade 6 of bisenone macrocycle 3 (Scheme 1). Conformational analysis 7 of 3 leads to a prediction wherein the resident stereocenters at C 2 , C 4 , and C 12 should mutually reinforce the desired stereochemical course of the reaction. This plan permits the convergent assembly of vinyl iodide Scheme 5. Transannular Cyclization Analysis

The polyketide synthase CTB1 is demonstrated to catalyze pyrone formation thereby expanding the known biosynthetic repertoire of thioesterase domains in iterative, non-reducing polyketide synthases.

The endophyte presence and diversity in natural populations of Poa alsodes were evaluated along a latitudinal transect from the southern distribution range in North Carolina to New York. Two distinct Epichloë hybrid taxa were identified from 23 populations. Each taxon could easily be distinguished by polymerase chain reaction (PCR) genotyping with primers designed to mating type genes and alkaloid biosynthesis genes that encode key pathway steps for ergot alkaloids, indole-diterpenes, lolines, and peramine. The most commonly found Epichloë taxon, Poa alsodes Taxonomic Group-1 (PalTG-1), was detected in 22 populations at high infection frequencies (72-100%), with the exception of one population at high elevation (26% infection). The second taxon, PalTG-2, was observed only in five populations in Pennsylvania constituting 12% of infected samples. Phylogenetic analyses placed PalTG-1 as an interspecific hybrid of E. amarillans and E. typhina subsp. poae ancestors, and it is considered a new hybrid species, which the authors name Epichloë alsodes. PalTG-2 is an intraspecific hybrid of two E. typhina subsp. poae ancestors, similar to E. schardlii from the host Cinna arundinacea, which the authors propose as a new variety, Epichloë schardlii var. pennsylvanica. Epichloë alsodes isolates were all mating type MTA MTB and tested positive for dmaW, easC, perA, and some LOL genes, but only the alkaloid N-acetylnorloline was detected in E. alsodes-infected plant material. Epichloë schardlii var. pennsylvanica isolates were all mating type MTB MTB and tested positive for perA, but peramine was not produced. Both E. alsodes and E. schardlii var. pennsylvanica appeared to have complete perA genes, but point mutations were identified in E. alsodes that would render the encoded perA gene nonfunctional.