Cloning and Characterization of an Environmental DNA-Derived Gene Cluster That Encodes the Biosynthesis of the Antitumor Substance BE-54017

Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, New York 10065, USA.
Journal of the American Chemical Society (Impact Factor: 11.44). 05/2011; 133(26):9996-9. DOI: 10.1021/ja2022653
Source: PubMed

ABSTRACT Soil is predicted to contain thousands of unique bacterial species per gram. Soil DNA libraries represent large reservoirs of biosynthetic diversity from which diverse secondary metabolite gene clusters can be recovered and studied. The screening of an archived soil DNA library using primers designed to target oxytryptophan dimerization genes allowed us to identify and functionally characterize the first indolotryptoline biosynthetic gene cluster. The recovery and heterologous expression of an environmental DNA-derived gene cluster encoding the biosynthesis of the antitumor substance BE-54017 is reported here. Transposon mutagenesis identified two monooxygenases, AbeX1 and AbeX2, as being responsible for the transformation of an indolocarbazole precursor into the indolotryptoline core of BE-54017.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Tryptophan, the most chemically complex and the least abundant of the 20 common proteinogenic amino acids, is a biosynthetic precursor to a large number of complex microbial natural products. Many of these molecules are promising scaffolds for drug discovery and development. The chemical features of tryptophan, including its ability to undergo enzymatic modifications at almost every atom in its structure and its propensity to undergo spontaneous, non-enzyme catalyzed chemistry, make it a unique biological precursor for the generation of chemical complexity. Here, we review the pathways that enable incorporation of tryptophan into complex metabolites in bacteria, with a focus on recently discovered, unusual metabolic transformations. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Chemistry & biology 03/2015; 22(3):317-328. DOI:10.1016/j.chembiol.2015.02.005 · 6.59 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Indolotryptoline natural products represent a small family of structurally unique chromopyrrolic acid derived antiproliferative agents. Like many prospective anticancer agents before them, the exploration of their potential clinical utility has been hindered by the limited information known about their mechanism of action. To study the mode of action of two closely related indolotryptolines (BE-54017, cladoniamide A), we selected for drug resistant mutants using a multidrug resistance-suppressed (MDR-sup) Schizosaccharomyces pombe strain. As fission yeast maintains many of the basic cancer-relevant cellular processes present in human cells, it represents an appealing model to use in determining the potential molecular target of antiproliferative natural products through resistant mutant screening. Full genome sequencing of resistant mutants identified mutations in the c and c' subunits of the proteolipid substructure of the vacuolar H+-ATPase complex (V-ATPase). This collection of resistance conferring mutations map to a site that is distant from the nucleotide-binding sites of V-ATPase and distinct from sites found to confer resistance to known V-ATPase inhibitors. Acid vacuole staining, cross-resistance studies and direct c/c' subunit mutagenesis all suggest that indolotryptolines are likely a structurally novel class of V-ATPase inhibitors. This work demonstrates the general utility of resistant mutant selection using MDR-sup S. pombe as a rapid and potentially systematic approach for studying the modes of action of cytotoxic natural products.
    Biochemistry 10/2014; DOI:10.1021/bi501078j · 3.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Indolotryptolines are bisindole natural products isolated from microbial and eDNA sources. Here we report the sequence of transformations that convert an indolocarbazole to the indolotryptoline cladoniamide through reconstruction of the four-enzyme cascade in E. coli. This cascade involves, first, conversion of an indolocarbozole to a C4c-C7a cis diol by ClaX1; second, N-methylation by ClaM1; third, rearrangement to the indolotryptoline scaffold by ClaX2; and fourth, installation of an O-methyl group by ClaM3. We furthermore elucidate the origins of minor cladoniamides D-G as the products of non-enzymatic, base-catalyzed opening of the succinimide ring of cladonimiades A-B. Overall, this work reveals the tenuous pathway indolocarbazole-derived metabolites must traverse as they are converted into indolotryptoline products and highlights the importance of non-enzymatic chemistry in generating bisindole diversity.
    ACS Chemical Biology 10/2014; 9(12). DOI:10.1021/cb500728h · 5.36 Impact Factor

Full-text (2 Sources)

Available from
Jun 4, 2014