Sample records for tyrocidine
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Troskie, Anscha Mari; Rautenbach, Marina; Delattin, Nicolas; Vosloo, Johan Arnold; Dathe, Margitta; Thevissen, Karin
2014-01-01
Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro. Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment. PMID:24752256
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Troskie, Anscha Mari; Rautenbach, Marina; Delattin, Nicolas; Vosloo, Johan Arnold; Dathe, Margitta; Cammue, Bruno P A; Thevissen, Karin
2014-07-01
Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro. Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Construction of hybrid peptide synthetases by module and domain fusions
Mootz, Henning D.; Schwarzer, Dirk; Marahiel, Mohamed A.
2000-01-01
Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min-1 and 2.1 min-1. The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides. PMID:10811885
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Construction of hybrid peptide synthetases by module and domain fusions.
Mootz, H D; Schwarzer, D; Marahiel, M A
2000-05-23
Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min(-1) and 2.1 min(-1). The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides.
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Cameron, Alan J; Edwards, Patrick J B; Harjes, Elena; Sarojini, Vijayalekshmi
2017-12-14
The d-Phe-Pro β-turn of the cyclic β-hairpin antimicrobial decapeptide tyrocidine A, (Tyrc A) was substituted with the d-Phe-2-aminobenzoic acid (2-Abz) motif in a synthetic analogue (1). The NMR structure of 1 demonstrated that compound 1 retained the β-hairpin structure of Tyrc A with additional planarity, resulting in approximately 30-fold reduced hemolysis than Tyrc A. Although antibacterial activity was partially compromised, a single Gln to Lys substitution (2) restored activity equivalent to Tyrc A against S. aureus, enhanced activity against two Gram negative strains and maintained the reduced hemeloysis of 1. Analysis by transmission electron microscopy (TEM) suggested a membrane lytic mechanism of action for these peptides. Compound 2 also exhibits nanomolar antifungal activity in synergy with amphotericin B. The d-Phe-2-Abz turn may serve as a tool for the synthesis of structurally predictable β-hairpin libraries. Unlike traditional β-turn motifs such as d-Pro-Gly, both the 2-Abz and d-Phe rings may be further functionalized.
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Lee, Sung G.; Lipmann, Fritz
1977-01-01
Dissociation of the multienzymes of tyrocidine synthesis by prolonged incubation of crude extracts of Bacillus brevis (Dubos strain, ATCC 8185) has yielded, on Sephadex G-100 chromatography, two fractions of amino acid activating subunits, a larger one of 70,000 daltons and a smaller one of 90,000 daltons; the latter was a complex consisting of the 70,000 dalton subunit and the pantetheine-carrying protein of about 20,000 daltons. When it dissociated, the intermediate enzyme, which activates three amino acids, contained two-thirds of the subunits in the 70,000 dalton and one-third in the 90,000 dalton fraction; the heavy enzyme, which activates six amino acids, contained five-sixths of the subunits in the former fraction and one-sixth in the latter. Both fractions showed ATP-PPi exchange with all amino acids that are activated by the respective polyenzymes. With proline as an example, the 70,000 dalton subunit exhibited a single low-affinity binding site, which should correspond to the peripheral thiol acceptor site, whereas the 90,000 dalton subunit showed both a low-affinity binding site and an additional high-affinity site for proline; the high-affinity site is attributed to the pantetheine present on the pantetheine-carrying protein, and suggests that amino acids are translocated from the peripheral SH to the pantetheine-carrying moiety during chain elongation. This was confirmed by the observation that the 90,000 dalton complex, when incubated with the light enzyme in the presence of phenylalanine and proline, produced DPhe-Pro dipeptide that cyclized into DPhe-Pro diketopiperazine, but the 70,000 dalton activating subunit, when similarly incubated, did not. After subunit dissociation, however, no further elongation occurred after the transfer from phenylalanine to proline. Images PMID:196286
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Tobin, M B; Kovacevic, S; Madduri, K; Hoskins, J A; Skatrud, P L; Vining, L C; Stuttard, C; Miller, J R
1991-01-01
Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete. Images PMID:1917855
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Peptide Epimerization Machineries Found in Microorganisms.
Ogasawara, Yasushi; Dairi, Tohru
2018-01-01
D-Amino acid residues have been identified in peptides from a variety of eukaryotes and prokaryotes. In microorganisms, UDP- N -acetylmuramic acid pentapeptide (UDP-MurNAc-L-Ala-D-Glu-meso-diaminopimelate-D-Ala-D-Ala), a unit of peptidoglycan, is a representative. During its biosynthesis, D-Ala and D-Glu are generally supplied by racemases from the corresponding isomers. However, we recently identified a unique unidirectional L-Glu epimerase catalyzing the epimerization of the terminal L-Glu of UDP-MurNAc-L-Ala-L-Glu. Several such enzymes, introducing D-amino acid resides into peptides via epimerization, have been reported to date. This includes a L-Ala-D/L-Glu epimerase, which is possibly used during peptidoglycan degradation. In bacterial primary metabolisms, to the best of our knowledge, these two machineries are the only examples of peptide epimerization. However, a variety of peptides containing D-amino acid residues have been isolated from microorganisms as secondary metabolites. Their biosynthetic mechanisms have been studied and three different peptide epimerization machineries have been reported. The first is non-ribosomal peptide synthetase (NRPS). Excellent studies with dissected modules of gramicidin synthetase and tyrocidine synthetase revealed the reactions of the epimerization domains embedded in the enzymes. The obtained information is still utilized to predict epimerization domains in uncharacterized NRPSs. The second includes the biosynthetic enzymes of lantibiotics, which are ribosome-dependently supplied peptide antibiotics containing polycyclic thioether amino acids (lanthionines). A mechanism for the formation of the D-Ala moiety in lanthionine by two enzymes, dehydratases catalyzing the conversion of L-Ser into dehydroalanine and enzymes catalyzing nucleophilic attack of the thiol of cysteine into dehydroalanine, was clarified. Similarly, the formation of a D-Ala residue by reduction of the dehydroalanine residue was also reported. The last type of machinery includes radical- S -adenosylmethionine (rSAM)-dependent enzymes, which catalyze a variety of radical-mediated chemical transformations. In the biosynthesis of polytheonamide, a marine sponge-derived and ribosome-dependently supplied peptide composed of 48 amino acids, a rSAM enzyme (PoyD) is responsible for unidirectional epimerizations of multiple different amino acids in the precursor peptide. In this review, we briefly summarize the discovery and current mechanistic understanding of these peptide epimerization enzymes.