Biosynthetic Pathways DOI: 10.1002/ange.201201445 A Sweet Origin for the Key Congocidine Precursor 4- Acetamidopyrrole-2-carboxylate** Sylvie Lautru,* Lijiang Song, Luc Demange, Thomas Lombs, HervØ Galons, Gregory L. Challis, and Jean-Luc Pernodet Pyrrole groups are found in nature in two primary metabolites (heme and tryptophan) and in a large variety of secondary metabolites (e.g. pyoluteorin, clorobiocin, congocidine, and prodigiosins). The diversity of these metabolites is mirrored in the multiple biosynthetic pathways leading to pyrrole groups. Six different biosynthetic pathways have been char- acterized to date, [1, 2] involving various primary metabolite precursors, such as amino acids (glycine, proline, serine, threonine, and tryptophan), [1–10] dicarboxylic acids (malonate, oxaloacetate, and succinate), [1, 2, 7, 8] or N-(5’-phosphoribosyl)- anthranilate. [1] Pyrrolamides are a family of natural products, synthesized by Streptomyces and related actinobacteria, that all contain one or more pyrrole-2-carboxamide moieties in their struc- ture. Most pyrrolamides, such as the well-characterized congocidine (1, also called netropsin; Figure 1) and distamy- cin, bind noncovalently in the DNA minor groove with some sequence specificity, [11] but pyrronamycin B has been sug- gested to bind DNA covalently. [12] This capacity to bind DNA confers on them a variety of biological activities, such as antiviral, antibacterial, antitumor, and anthelmintic activities, but also renders them too toxic for clinical use. Nonetheless, because molecules binding to DNA at specific positions can manipulate the expression of genes involved in various diseases (such as cancer), congocidine and distamycin have prompted the synthesis of many structurally related mole- cules that bind in the minor groove of DNA at various defined sequences. [13] The nature and biosynthetic origin of the pyrrolamide pyrrole precursor are unknown. A retrobiosynthetic analysis of pyrrolamide structures suggests 4-aminopyrrole-2-carbox- ylate as the potential pyrrole precursor common to all pyrrolamides. However, this remains to be established and no biosynthetic pathway has been reported for the synthesis of this molecule. We report herein that 4-acetamidopyrrole-2- carboxylate (10) is the true precursor of congocidine and propose for this compound a biosynthetic pathway starting from N-acetylglucosamine-1-phosphate (2), involving carbo- hydrate metabolizing enzymes, and differing entirely from known pyrrole biosynthetic pathways. We recently reported the identification, analysis, and heterologous expression of the first pyrrolamide gene cluster ; the cgc gene cluster directs congocidine biosynthesis in Streptomyces ambofaciens . [14] Sequence analyses of the pro- teins encoded by the cgc cluster led us to propose a pathway for congocidine biosynthesis involving a noncanonical non- ribosomal peptide synthetase (NRPS) and three putative precursors: guanidinoacetate, 3-aminopropionamidine, and 4- aminopyrrole-2-carboxylate. However, the biosynthetic ori- gins of these precursors could not easily be inferred from the analysis of the cgc gene cluster. In particular, we could not identify any gene encoding homologues of known pyrrole biosynthetic enzymes, suggesting that the pyrrole groups in pyrrolamides could be synthesized through an entirely new pathway. The cgc18 gene encodes an NRPS module that has been proposed to recognize 4-aminopyrrole-2-carboxylate and catalyze its ATP-dependent activation as the corresponding peptidyl carrier protein (PCP)-bound thioester. [14] To inves- tigate whether 4-aminopyrrole-2-carboxylate is indeed a pre- cursor of congocidine, we used HPLC to compare the profile of metabolites in the culture supernatants of the “wild type” SPM110 Streptomyces ambofaciens strain and the CGCA004 mutant strain, in which cgc18 has been disrupted. [14] A new compound accumulated in the culture supernatant of CGCA004 (retention time = 12.3 min, Figure 2 a), which we expected to be 4-aminopyrrole-2-carboxylate. This metabo- lite peak was not detected in the CGCA004 chromatogram of our previous report [14] and on average, it was detected in approximately half of the cultures of strains deleted in cgc [*] Dr. S. Lautru, Dr. J.-L. Pernodet Institut de GØnØtique et Microbiologie UniversitØ Paris-Sud, CNRS, UMR8621, 91405 Orsay (France) E-mail: sylvie.lautru@igmors.u-psud.fr Dr. L. Song, Prof. G. L. Challis Department of Chemistry, University of Warwick, Coventry CV4 7AL (UK) Dr. L. Demange, Prof. H. Galons Laboratoire de Chimie et Biochimie Pharmacologiques et Toxico- logiques UMR 8601 CNRS UniversitØ Paris Descartes, Sorbonne Paris CitØ, UFR BiomØdicale des Saints Pres 45 rue des Saints Pres, 75006 Paris (France) T. Lombs UMR 8638 CNRS-Paris Descartes, FacultØ des Sciences Pharma- ceutiques et Biologiques 4 av. de l’Observatoire, 75006 Paris (France) [**] We would like to thank Dr. Christophe Corre for helpful discussions, Dr. Laurent Micouin for his help with the synthesis of the 4- acetamidopyrrole-2-methanol, and Dr. Cyrille Kouklovsky, GØraldine Le Goff, and Dr. Robert Thaï for their help with mass spectrometry. This work was supported in part by the European Union through the Integrated Project ActinoGEN (CT-2004-0005224) and the reinte- gration grant BOPA (CT-2005-029154), by the Pôle de Recherche et d’Enseignement SupØrieur UniverSud Paris and by the Bettencourt Schueller Foundation. Supporting information for this article (experimental details) is available on the WWW under http://dx.doi.org/10.1002/anie. 201201445. . Angewandte Zuschriften 7572  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2012, 124, 7572 –7576