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Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases.

Albicidin is produced by the sugarcane pathogenic bacterium Xanthomonas albilineans and is a potent antibiotic specifically targeting the bacterial DNA gyrase with an IC50 value in the nanomolar range. The structure of albicidin, which has remained unsolved for more than three decades since its first description, has been solved by a combination of extensive NMR and HR-MS/MS experiments. An inherent problem for the structure elucidation was the low amount of the albicidin production in cell culture. The low production forced us to cultivate several hundred liters in order to purify a few milligrams required for NMR experiments. In comparison, LC-HR-MS/MS has the great advantage of several orders of magnitude and high sensitivity. Thus, this approach requires much less material. But more importantly, purification of compounds is not necessary for structural analysis. Due to the application of targeted LC-MS/MS experiments, we were able to propose the structure of N-terminal carba- S158 moylated and b-methoxylated albicidin derivatives. Both of them are currently chemically synthesized and studied in a medicinal chemistry campaign. In order to search for more pharmaceutically relevant albicidin derivatives, we applied non-targeted LC-HR-MS/MS approaches combined with mass spectral networking, a recently introduced approach for non-targeted MS/MS data-analysis. However, as for the structural elucidation of albicidin, the even lower concentrated derivatives resulted in difficulties to obtain useful MS/ MS spectra. Since we typically operate the mass spectrometer in data-dependent acquisition mode (DDA), it inherently selects high abundant compounds of cell extracts for MS/ MS scans and possibly ignore low abundant compounds. The same goes for the network analysis which is based on similarity of MS/MS spectra and may not take potential ion species into account. To overcome this problem, we implemented a bioactivity-guided fractionation by solid phase extraction and semi-preparative HPLC. Fractions with antibacterial activity were subsequently submitted to several LC-MS/MS runs in DDA mode with shifted survey scan windows (stitched DDA). Besides the increased amount of MS/MS events per mass range, narrowing down the m/z range of the survey scan also increased the dynamic range of the orbitrap analyzer, which enabled us to significantly increase the method's sensitivity. Finally, we were able to detect a whole series of new albicidin derivatives and to propose their structures based on exact mass determination and MS/MS fragmentation patterns. These new compounds provide great opportunities in lead optimization of albicidin as a new anti-infective drug. Apart from the pharmaceutical relevance, some of the identified by-products give us important insights into the formation of albicidin and its intriguing biosynthetic machinery.

Albicidin is a potent DNA gyrase inhibitor produced by the sugarcane pathogenic bacterium Xanthomonas albilineans. It blocks the d ifferentiation of chloroplasts, resulting in appearance of narrow white stripes on sugarcane leaves that are characteristic of leaf scald d isease. Albicidin targets the bacterial gyrase by a mechanism that is different from the one of any other known DNA gyrases inhibitors [l]. It exhibits antibacterial activity at nanomolar concentrations against Escherichia coli and to a lower extent aga inst numerous human pathogenic bacteria [2]. A decade of intense work was necessary to decipher a lbicid in's biosynthetic pathway and to elucidate its astonishing never-seen-before structure. Albicidin is produced by a hybrid PKS/NRPS system. Such ribosome-independent systems cons ist of modular megasynthetases which operate in an assembly-line fashion to activate, modify and link mostly unusual aminoacid build ing blocks, fina lly resulting in complex bioactive peptide-like molecules. The structure of albicidin, which was predicted by fo rmer in silico sequence a nalyses of its PKS/ NRPS gene cluster [3], was ascertained by means of mass spectrometry and NMR spectroscopy. We demonstrated that a lbicidin exhibits a linear polyaromatic penta-peptidic structure containing the rare aminoacids para-aminobenzoate a nd cyanoalanine [4]. Insights into biosynthesis and structure of natural a nalogues of al bicidin will also be presented [5]. The determination of the structure of albicidin allowed the development of a protocol for the chemical synthesis of this complex molecule [6]. Consequently, new research, such as structureactivity relationship studies, will now be possible.

Albicidin is a potent DNA gyrase inhibitor produced by the sugarcane pathogenic bacterium Xanthomonas albilineans. As such, this molecule blocks the differentiation of chloroplasts, resulting in appearance of narrow white stripes on sugarcane leaves that are characteristic of leaf scald disease. Albicidin targets the bacterial gyrase by a mechanism that is different from the one of other DNA gyrases inhibitors like coumarins or quinolones [1]. It also exhibits antibacterial activity at nanomolar concentrations against Escherichia coli and to a lower extent against numerous Gram-negative and -positive human pathogenic bacteria [2].A decade of intense work was necessary to decipher albicidin's biosynthetic pathway and to elucidate its astonishing never-seen-before structure. Albicidin is produced by a hybrid PKS/NRPS (polyketide synthase/non ribosomal peptide synthetase) system. Such ribosome-independent systems consist of modular megasynthetases which operate in an assembly-line fashion to activate, modify and link mostly unusual aminoacid building blocks, finally resulting in complex bioactive peptide-like molecules. The structure of albicidin, which was predicted by former in silico sequence analyses of its PKS/NRPS gene cluster [3], was ascertained by means of mass spectrometry and nuclear magnetic resonance spectroscopy. We were able to demonstrate that albicidin exhibits a linear polyaromatic penta-peptidic structure containing the rare aminoacids para-aminobenzoate and cyanoalanine [4]. The determination of the structure of albicidin allowed the development of a protocol for the chemical synthesis of this complex molecule. Consequently, new research, such as structure-activity relationship studies, will now be possible [5].New insights into biosynthesis pathway and structural determination for albicidin will be presented. (Texte intégral)

Albicidin is a potent inhibitor of prokaryotic DNA gyrase, produced by the sugarcane pathogenic bacterium Xanthomonas albilineans. The structure of albicidin remained unclear for more than three decades after its first description by Birch et al.[1]. Most recently we were able to solve the unknown structure, revealing a unique polyaromatic oligopeptide mainly composed of p-amino benzoic acids[2]. With the knowledge of the structure we performed furthermore the total synthesis of albicidin [3].In the bachelor thesis we show the in-vitro study of the non-ribosomal albicidin assembly line, which provide further insights into the biosynthetic machinery of albicidin. Together with our bioinformatic investigations we were able to propose a comprehensive biochemical assembly, expanding the non-ribosomal code of adenylation domains with p-amino benzoicacid derivatives.

Albicidins, produced by the sugarcane pathogenic bacterium Xanthomonas albilineans, are potent inhibitors of the bacterial DNA gyrase with Ic50values in nM ranges [1].The structure of albicidins remained unclear for more than three decades after their first description by Birch et al. [2]. After the identification and sequencing of three gene islands, responsible for the albicidin biosynthesis, a PKS-NRPS hybrid, build up by three enzymes, Alb01, Alb05and Alb09 was proposed for the albicidin assembly [3, 4].Most recently we were able to solve the hitherto unknown structure of the main albicidin [5,6] and post NRPS modified derivatives, revealing a unique polyaromatic oligopeptide mainly composed of p-amino benzoic acids. Our in-vitro studies of the non-ribosomal albicidin assembly line and tailoring enzymes provide detailed insights into the biosynthetic machinery of albicidins. Together with our bioinformatic investigations we are able to propose a comprehensive biochemical assembly, expanding the non-ribosomal code of adenylation domains with p-amino benzoic acid derivatives. Furthermore our study revealed a new type of dehydratase domain responsible for the in situ formation and incorporation of cyano-alanine [6]. (Texte intégral)

The present invention relates to a antibiotically active compounds characterized by general formula (I), wherein X1, BB, BC, BD, BE and X2 are building blocks with D1, D2, D3, D4 or D5 being linkers which comprise carbon, sulphur, nitrogen, phosphor and/or oxygen atoms and which are covalently connecting the moities BA and BB, BB and BC, BC and BD, BD and BE and BE and BF, respectively, and wherein in particular the building block BC comprises an amino acid derivative. The invention relates further to said compounds for use in a method of treatment of diseases, in particular for use in a method of treatment of bacterial infections.

De nombreuses bactéries et de nombreux champignons utilisent la voie non ribosomale pour produire des peptides ou d'autres petites molécules. Cette voie, nommée NRPS pour " non-ribosomal peptide synthesis ", fait appel à des mégaenzymes modulaires qui catalyse nt l'assemblage de peptides à partir d'acides aminés protéinogéniques ou non protéinogéniques. Les petites molécules synthétisées par cette voie peuvent intervenir dans des processus très variés tels que "adaptation aux environnements défavorables, la communication ou la compétition avec d'autres microorganismes dans leur habitat naturel, ou encore l'interaction avec un hôte. Xanthomonas albilineans est l'agent causal de "échaudure des feuilles, une des principales maladies bactériennes de la canne à sucre. Le séquençage de son génome a conduit à la découverte d'un nouveau locus NRPS, appelé META-B, qui est également présent dans le génome de souches appartenant à Xanthomonas oryzae ou Xanthomonas /rans/ucens. Les analyses in silico indiquent que ce locus ne ressemble à aucun autre locus décrit à ce jour et que chaque souche séquencée produit un peptide META-B différent. La présence du locus META-B chez trois espèces de Xanthomonas phylogénétiquement distantes et associées à des plantes monocotylédones suggère que la nouvelle famille de petites molécules produites par ce locus pourrait jouer un rôle dans les interactions plante-bactérie. Un protocole permettant de surproduire et d' isoler le peptide META-B chez la souche GPE PC73 de X. abilineans a été développé. Deux approches sont actuellement mises en oeuvre pour trouver la fonction de ce peptide META-B. La première est basée sur l'étude du peptide META-B purifié (tests in vitro pour évaluer les activités antibactériennes ou antifongiques ou pour mettre en évidence un rôle dans les interactions avec la canne à sucre). La seconde est basée sur l'étude de mutants affectés dans l'expression de gènes du locus META-B (expérimentations sur canne à sucre). (Texte intégral)

International audience; Various bacteria use non-ribosomal peptide synthesis (NRPS) to produce peptides or other small molecules. These molecules exhibit broad structural diversity and display biological activities that range from adaptation to unfavorable environments, communication or competition with other microorganisms in their natural habitat, or even to action as virulence factors. Conserved features within the NRPS machinery allow the type, and sometimes even the structure, of the synthesized polypeptide to be predicted. Thus, bacterial genome mining via in siIico analyses of NRPS genes offers an attractive opportunity to uncover new bioactive non-ribosomally synthesized peptides. To date, the only known small molecule synthesized by NRPS in the genus Xanthomonas is albicidin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald-a lethal disease of sugarcane. In silica analysis of available genomic sequences of Xanthomonas strains led to the discovery of a novel NRPS gene cluster called META-B which doesn't resemble to any gene cluster de- scribed to date. This NRPS gene cluster occurs in (i) X. albilineans, (ii) two pathovars of Xanthomonas oryzae which are the causal agents of two agronomically important diseases of rice (bacterial leaf blight caused by X. oryzae pv. oryzae and bacterial leaf streak caused by X. oryzae pv. oryzicola), and (iii) Xanthomonas translucens , the causal agent of the bacterial leaf streak of wheat. Interestingly, the NRPS gene cluster META-B seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement in plant-bacteria interactions. (Résumé d'auteur)

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich. This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. The final biosynthetic step towards plantazolicin (PZN) comprises Nα,Nα-arginyl methyltransferase (PznL) mediated N-terminal bismethylation. We show that PznL processes truncated desmethyl-plantazolicin analogues, but only those with an N-terminal guanidine side chain derived from arginine. PznL specificity, which is narrow, depends on the side chain of the N-terminal amino acid linked to an azole, and not so much on the number of azoles.

Background: To date, the only known small molecule synthesized by non-ribosomal peptide synthesis (NRPS) in the genus Xanthomonas is the phytotoxin albicidin produced by Xanthomonas albilineans, the causal agent of sugarcane leaf scald disease. Recent analyses of available genomic sequences indicated that X. albilineans, as well as Xanthomonas oryzae and Xanthomonas translucens, possess a novel NRPS gene cluster called META-B whose architecture is strainspecific (each sequenced strain is predicted to synthesize a different peptide) and doesn´t resemble to any NRPS gene cluster described to date. Because the META-B NRPS gene cluster is specific to three phylogenetically distant species of Xanthomonas associated with monocotyledonous plants, we assume a putative involvement of the new family of small molecules produced by this gene cluster in plant-bacteria interactions. Objectives: We aimed at isolating and characterizing the small molecule encoded by the META-B NRPS gene cluster of X. albilineans in order to study its putative involvement in plant-bacteria interactions. Methods: We developed a META-B overproducing X. albilineans strain by over-expressing the AraC transcriptional regulator which is present within the META-B NRPS gene cluster. HPLC profiles obtained from cultures of wild type vs the AraC over-expressing strain were compared. Peaks with significantly increased amplitude in the HPLC profile from the AraC over-expressing strain were collected and their content analyzed by mass spectrometry. Conclusions: We identified a peptide with a nominal molecular mass > 2 kDa and with an amino acid sequence (MS/MS experiments) which excellently matches the one predicted by A-domain specificities of META-B NRPS genes. (Texte intégral)

Xanthomonas is a large genus of Gram-negative bacteria that cause disease in hundreds of plant species. To date, the only known small molecule synthesized by non-ribosomal peptide synthesis (NRPS) in this genus is albicidin produced by Xanthomonas albilineans. The DNA gyrase inhibitor albicidin is not only an important virulence factor but also a possible lead structure for novel antibiotics. This study aims to estimate the biosynthetic potential of Xanthomonas spp. by in silico analyses of NRPS genes with unknown function recently identified in the sequenced genomes of X. albilineans and related species of Xanthomonas. We performed in silico analyses of NRPS genes present in all published genome sequences of Xanthomonas spp., as well as in unpublished draft genome sequences of Xanthomonas oryzae pv. oryzae strain BAI3 and Xanthomonas spp. strain XaS3. The most unexpected result of these analyses is that these two latter strains, together with X. albilineans strain GPE PC73 and X. oryzae pv. oryzae strains X8-1A and X11-5A, possess novel NRPS gene clusters. Furthermore, these Xanthomonas spp. strains share related NRPS-associated genes such as those required for the biosynthesis of non-proteinogenic amino acids or for the secretion of peptides. In silico prediction of peptide structures according to the NRPS architecture accounts for eight different peptides, each specific to its producing strain. Interestingly, these eight peptides cannot be assigned to any known gene cluster or related to known compounds from natural product databases. PCR screening of a collection of 94 plant pathogenic bacteria indicates that these novel NRPS gene clusters are specific to the genus Xanthomonas and are also present in Xanthomonas transluscens and X. oryzae pv. oryzicola. Further genome mining revealed (i) novel NRPS genes shared by Xanthomonas spp. strains GPE PC73 and XaS3 with the plant-associated bacterium Bradyrhizobium spp. strain BTAi and (ii) novel NRPS genes specific to X. oryzae pv. oryzicola or Xanthomonas sacchari. This study revealed the significant potential of the genus Xanthomonas of producing new non-ribosomally synthesized peptides. Interestingly, this biosynthetic potential seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement of new non-ribosomally synthesized peptides in plant-bacteria interactions.