The enediynes exemplify nature's ingenuity. We have cloned and characterized the biosynthetic locus coding for perhaps the most notorious member of the nonchromoprotein enediyne family, calicheamicin. This gene cluster contains an unusual polyketide synthase (PKS) that is demonstrated to be essential for enediyne biosynthesis. Comparison of the calicheamicin locus with the locus encoding the chromoprotein enediyne C-1027 reveals that the enediyne PKS is highly conserved among these distinct enediyne families. Contrary to previous hypotheses, this suggests that the chromoprotein and nonchromoprotein enediynes are generated by similar biosynthetic pathways.
Genome analysis of actinomycetes has revealed the presence of numerous cryptic gene clusters encoding putative natural products. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we demonstrate the use of a high-throughput genome scanning method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover biosynthetic pathways encoding enediyne antitumor antibiotics in a variety of actinomycetes. Comparative analysis of five biosynthetic loci representative of the major structural classes of enediynes reveals the presence of a conserved cassette of five genes that includes a novel family of polyketide synthase (PKS). The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring structure (or "warhead") found in all enediynes. Genome scanning analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.
The enediyne antitumor antibiotics are appreciated for their novel molecular architecture, their remarkable biological activity and their fascinating mode of action and many have spawned considerable interest as anticancer agents in the pharmaceutical industry. Of equal importance to these astonishing properties, the enediynes also offer a distinct opportunity to study the unparalleled biosyntheses of their unique molecular scaffolds and what promises to be unprecedented modes of self-resistance to highly reactive natural products. Elucidation of these aspects should unveil novel mechanistic enzymology, and may provide access to the rational biosynthetic modification of enediyne structure for new drug leads, the construction of enediyne overproducing strains and eventually lead to an enediyne combinatorial biosynthesis program. This article strives to compile and present the critical research discoveries relevant to the clinically most promising enediyne, calicheamicin, from a historical perspective. Recent progress, particularly in the areas of biosynthesis, self-resistance, bio-engineering analogs and clinical studies are also highlighted.
A universal PCR method for the rapid amplification of minimal enediyne polyketide synthase (PKS) genes and the application of this methodology to clone remaining prototypical genes from producers of structurally determined enediynes in both family types are presented. A phylogenetic analysis of the new pool of bona fide enediyne PKS genes, consisting of three from 9-membered producers (neocarzinostatin, C1027, and maduropeptin) and three from 10-membered producers (calicheamicin, dynemicin, and esperamicin), reveals a clear genotypic distinction between the two structural families from which to form a predictive model. The results from this study support the postulation that the minimal enediyne PKS helps define the structural divergence of the enediyne core and provides the key tools for generating enediyne hybrid genes͞molecular scaffolds; by using the model, a classification is also provided for the unknown enediyne PKS genes previously identified via genome scanning.
Nature continues to be the inspiration for most pharmaceutical drug leads, and given the synthetic challenge posed by many complex secondary metabolites, the emerging field of combinatorial biosynthesis has become a rich new source for modified non-natural scaffolds. 1 Yet, many naturally occurring bioactive secondary metabolites possess unusual carbohydrate ligands, which serve as molecular recognition elements critical for biological activity. 2 Without these essential sugar attachments, the biological activities of most clinically important secondary metabolites are either completely abolished or dramatically decreased. We and others have demonstrated that glycosyltransferases, responsible for the final glycosylation of certain secondary metabolites, show a high degree of promiscuity toward the nucleotide sugar donor. 3,4 These discoveries have opened the door to the possibility of manipulating the corresponding biosynthetic pathways for modifying the crucial glycosylation pattern of natural, or non-natural, secondary metabolite scaffolds in a combinatorial fashion. To date, the genetic manipulation of the carbohydrate appendage for any given metabolite has been limited to alterations and/or knock-outs of the small subset of genes required to construct and attach each desired carbohydrate moiety. However, a significant expansion of the saccharide structure diversity obtained by these methods might be accomplished via the recruitment and collaborative action of sugar genes from a variety of biosynthetic pathways to construct composite clusters with the potential to make and attach non-natural sugars.To test this possibility, we selected the Streptomyces Venezuelae methymycin/pikromycin gene cluster as the parent system and a gene from the calicheamicin biosynthetic gene cluster (from Micromonospora echinospora spp. Calichensis) as the foreign collaborator gene. The parent cluster encodes the biosynthetic enzymes for methymycin (1), neomethymycin (2), pikromycin (3), and narbomycin (4), all of which are macrolides containing desosamine (5) as the sole sugar component crucial for antibiotic activity. 5 Eight open reading frames (desI-desVIII) in this cluster have been assigned as genes involved in desosamine biosynthesis (Scheme 1). The antitumor agent calicheamicin (6) contains four unique sugars crucial to tight DNA binding (K a ≈ 10 6 -10 8 ), one of which (9) is derived from 4-amino-4,6-dideoxyglucose (8) and is part of the unusually restricted N-O connection between sugars A and B (Scheme 2). 6 Compound 8 is anticipated to be derived from the corresponding 4-ketosugar 7 via a transamination reaction, and recent work has led to the assignment of a gene (calH) as encoding the desired C-4 aminotransferase (Scheme 2). 7 Interestingly, the proposed substrate for CalH, 7, is also an intermediate in the desosamine pathway and is expected to exist Thamchaipenet, A.; Gustafsson, C.; Fu, H.; Betlach, M.; Betlach, M.; Ashley, G. Targeted deletion/disruption of the desVI, desV, or desI genes in the methymycin/pikromycin...
Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins - together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster - show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the L-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.