Tyrocidine Synthesis Research Articles

Synthesis of Tyrocidine A and Its Analogues by Spontaneous Cyclization in Aqueous Solution

[reaction: see text] Head-to-tail cyclization of peptides is a multistep process involving tedious C-terminal activation and side chain protection. Here we report a facile, quantitative cyclization method in aqueous ammonia solution for the total syntheses of the cyclic decapeptide antibiotic Tyrocidine A and its analogues from their fully deprotected linear thioester precursors on a solid support. This novel aqueous method is conformation-dependent and may be applicable to syntheses of other natural cyclic peptides.

Induction of sporulation in Bacillus brevis. 2. Dependence on the presence of the peptide antibiotics tyrocidine and linear gramicidin.

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2-amino-3-hydroxy-3-phenylpropanoic acid [Mach, B., Reich, E., and Tatum, E. L. (1963) Proc. Natl Acad. Sci. USA, 50, 175-181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L-tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen-limited cultures. When either tyrocidine or linear gramicidin was added together with L-tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L-tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics.

Isolation of amino acid activating subunit-pantetheine protein complexes: Their role in chain elongation in tyrocidine synthesis

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-PP(i) 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.

ChemInform Abstract: STUDIES OF PEPTIDE ANTIBIOTICS. XXXIV. SYNTHESES OF TYROCIDINE A AND ITS ANALOGS CONTAINING GLYCINE

AbstractKondensation der ‐ durch schrittweisen Aufbau nach üblichen Methoden erhältlichen ‐ Komponenten (Ia) und (II), Überführung des gebildeten Esters (IIIa) über das Hydrazid (IVa) in das Azid, Cyclisierung und Schutzgruppenabspaltung liefern Tyrocidin A (Va).

Studies of Peptide Antibiotics. XXXIV. Syntheses of Tyrocidine A and Its Analogs Containing Glycine

Abstract The synthesis of Tyrocidine A (TA), an antibiotic cyclic decapeptide, was achieved by a revised conventional method. Two analogs of TA, 6-glycine-TA and 7-glycine-TA, were synthesized by a similar method to investigate the contribution of l-phenylalanine6 and d-phenylalanine7 residues in TA to its antibacterial activity. The properties of synthetic TA were identical to natural TA. The antibacterial activities of the two analogs were weaker when compared with TA; the activity of 6-glycine-TA being weaker than 7-glycine-TA. The three cyclic peptides synthesized were subjected to droplet countercurrent chromatography (DCCC) and optical rotatory dispersion (ORD), and the elucidation of the difference in the activities between the two analogs was followed with DCCC and ORD.

Induction of specific amino acid binding proteins preceding onset of tyrocidine synthesis in Bacillus brevis

AbstractA report on the induction of amino acid binding proteins prior to or together with the formation of enzymes for the biosynthesis of tyrocidine by Bacillus brevis.

The relation between sporulation and the induction of antibiotic synthesis and of amino acid uptake in Bacillus brevis.

The induction and localization of tyrocidine-synthesizing enzymes is shown to be parallel, during growth of Bacillus brevis (ATCC 8185, American Type Culture Collection, Rockville, Md.), with the induction of uptake of constitutive amino acids and of components of pantetheine, a coenzyme of tyrocidine synthesis. Antibiotic synthesis appears at the end of logarithmic growth when the first soluble enzymes may be obtained from homogenates. During this period, binding proteins for metabolite uptake were isolated by intensive sonication which, when studied by chromatography, were identified by the appearance of low molecular weight fractions binding the radioactively marked metabolites; their induction was prevented by addition of rifampicin. The major purpose of this study was a comparison of antibiotic production and sporulation, the progress of which was followed by electron microscopy. The onset of tyrocidine synthesis and metabolite uptake coincided with the appearance of septum formation indicating that sporulation had progressed to stage II. With the progress of spore encapsulation, the tyrocidine production migrated from the soluble fraction into the forespore, terminating with the separation of forespores from the sporangium membrane. The resulting concentration of antibiotic in the forespore may indicate its function in sporulation, the nature of which, however, was not explored.

Isolation of a peptidyl-pantetheine-protein from tyrocidine-synthesizing polyenzymes.

The polyenzyme complex responsible for the synthesis of tyrocidine in Bacillus brevis (ATCC 8185) was found to contain 4'-phosphopantetheine, which appeared to be connected with the production of growing peptide chains. Confirmation of this assumption has now been obtained by purifying from bacterial lysates a polyenzyme-dissociation product; this was labeled with [(14)C]pantothenic acid and peptide chains containing tritiated amino acids, and had a molecular weight of 17,000. To obtain these results, organisms were grown udner conditions favorable for incorporation of radioactive pantothenic acid into tyrocidine-synthesizing enzymes. A crude lysate of the [(14)C]pantothenic acid-labeled organisms was preincubated with the tritiated amino acids to form enzyme-bound growing peptide chains. The doubly labeled fragments were purified from the polyenzyme-dissociation products produced by prolonged lysis. In a second set of experiments, the three enzymes responsible for tyrocidine synthesis, including the two polyenzymes containing pantetheine, were purified and incubated with radioactive amino acids and ATP to form polyenzyme-bound peptide chains. Thereupon, a Triton X-100 extract of the 20,000 x g fraction of crude homogenate was added to dissociate the purified polyenzymes. The dissociation products were purified and yielded, on dodecyl sulfate gel electrophoresis, peptidyl-marked products ranging in molecular weight from 90,000 to 17,000, the latter being most abundant. Electrophoresis of analogous preparations after preincubation with higher concentrations of dodecyl sulfate and dithiothreitol at 100 degrees yielded a single product of 17,000 molecular weight, indicating that the larger molecular weight fractions were aggregates thereof.

Purification of the polyenzymes responsible for tyrocidine synthesis and their dissociation into subunits.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPurification of the polyenzymes responsible for tyrocidine synthesis and their dissociation into subunitsSung G. Lee, Robert Roskoski, Jr., Karl Bauer, and Fritz LipmannCite this: Biochemistry 1973, 12, 3, 398–405Publication Date (Print):January 1, 1973Publication History Published online1 May 2002Published inissue 1 January 1973https://doi.org/10.1021/bi00727a006RIGHTS & PERMISSIONSArticle Views40Altmetric-Citations44LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Studies of Peptide Antibiotics. XXL Synthesis of Tyrocidine C

Abstract A cyclic decapeptide, cyclo-(l-Trp-d-Trp-l-Asn-l-Gln-l-Tyr-l-Val-l-Orn-l-Leu-d-Phe-l-Pro-), having the amino acid sequence of natural tyrocidine C has been synthesized. Chemical and biological properties of this product have been described. An antibiological activity of the product toward Gram positive microorganisms was found to be nearly the same degree as that of tyrocidine B.

Synthesis of tyrocidine C

Synthesis of tyrocidine C

Biosynthesis of tyrocidine by a cell-free enzyme system of Bacillus brevis ATCC 8185 : I. Preparation of partially purified enzyme system and its properties

1. A tyrocidine-synthesizing system was partially purified from the crude extract of Bacillus brevis by ammonium sulfate fractionation, protamine sulfate treatment followed by another ammonium sulfate fractionation to a specific activity of about 20 mμmoles of tyrocidine formation per h per mg of protein. The partially purified system absolutely required Mg 2+ (or Mn 2+), ATP and the tyrocidine constituent amino acids for its activity. The optimum conditions for tyrocidine synthesis were found to be 2 mM Mg 2+ (or Mn 2+), 4 mM ATP, and pH 7.8 in the presence of 0.2–0.4 mM constituent amino acids. 2. AMP was formed from ATP in the process of tyrocidine synthesis and 1 molecule of ATP was required for the formation of one peptide bond. 3. The partially purified system catalyzed ATP-PP i exchange reactions in the presence of glycine, l-alanine, l-lysine and l-isoleucine, besides 9 tyrocidine constituent l-amino acids. The other non-constituent amino acids of tyrocidine did not promote the exchange reaction. 4. Column chromatography on DEAE-cellulose separated the partially purified system into two distinct components, Components I and II, both of which were required for tyrocidine synthesis.

Synthesis of tyrocidine A.

Synthesis of tyrocidine A.

EXPERIMENTS ON THE MECHANISM OF GRAMICIDIN AND TYROCIDINE SYNTHESIS IN CELL-FREE PREPARATIONS ON BACILLUS BREVIS.

EXPERIMENTS ON THE MECHANISM OF GRAMICIDIN AND TYROCIDINE SYNTHESIS IN CELL-FREE PREPARATIONS ON BACILLUS BREVIS.

Biosynthesis of gramicidin and tryocidine in the Dubos strain of Bacillus brevis. I. Experiments with growing cultures.

Okuda, Kiyoshi (Department of Biochemistry and Biophysics, University of Hawaii, Honolulu), Gordon C. Edwards, and Theodore Winnick. Biosynthesis of gramicidin and tyrocidine in the Dubos strain of Bacillus brevis. I. Experiments with growing cultures. J. Bacteriol. 85:329-338. 1963.-A simple chromatographic method was developed for the isolation of gramicidin and tyrocidine from tyrothricin of Bacillus brevis. A Tryptone-yeast extract-glucose medium containing mineral salts gave the best yields of peptides in a stationary culture of the organism. The incorporation of suitable C(14)-labeled amino acids into gramicidin and tyrocidine was studied Several analogues of tyrocidine amino acids (beta-hydroxyglutamic acid, pipecolic acid, beta-2-thienylalanine, p-fluorophenylalanine, and phenyl glycine) selectively reduced tyrocidine synthesis, when added to the nutrient medium. At the same time, the production of gramicidin was augmented. Growth and protein synthesis were not affected. Two analogues in isotopic form, beta-2-thienylalanine and isoleucine, were shown to give rise to high degrees of labeling in tyrocidine.