Tyrocidine Biosynthesis Research Articles

Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases

We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490nm of the l-Trp–hydroxamate–Fe3+ complex and the formation of l-Trp–l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.

Macrolactamization of Glycosylated Peptide Thioesters by the Thioesterase Domain of Tyrocidine Synthetase

The 35 kDa thioesterase (TE) domain excised from the megadalton tyrocidine synthetase (Tyc Syn) retains autonomous capacity to macrocyclize peptidyl thioesters to D-Phe1-L-Leu10-macrolactams. Since a number of nonribosomal peptides undergo O-glycosylation events during tailoring to gain biological activity, the Tyc Syn TE domain was evaluated for cyclization capacity with glycosylated peptidyl-S-NAC substrates. First, Tyr7 was replaced with Tyr(beta-D-Gal) and Tyr(beta-D-Glc) as well as with Ser-containing beta-linked D-Gal, D-Glc, D-GlcNAc, and D-GlcNH2, and these new analogs were shown to be cyclized with comparable kcat/Km catalytic efficiency. Similarly, Gal- or tetra-O-acetyl-Gal-Ser could also be substituted at residues 5, 6, and 8 in the linear decapeptidyl-S-NAC sequences and cyclized without substantial loss in catalytic efficiency by Tyc Syn TE. The cyclic glycopeptides retained antibiotic activity as membrane perturbants in MIC assays, opening the possibility for library construction of cyclic glycopeptides by enzymatic macrocyclization.

The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains.

The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.

Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis.

Tyrocidine is a cyclic decapeptide antibiotic which is produced and secreted by stationary-phase cells of the sporeforming bacterium Bacillus brevis. We identified the promoter for the B. brevis structural gene (tycA) for tyrocidine synthetase I, the enzyme catalyzing the first step in tyrocidine biosynthesis, and studied its regulation in cells of B. brevis and Bacillus subtilis. Transcription from the tycA promoter was induced at the end of the exponential phase of the growth cycle in B. brevis cells growing in sporulation medium. To study the regulation of tycA in B. subtilis, we constructed a derivative of the B. subtilis bacteriophage SP beta containing a transcriptional fusion of the tycA promoter to the lacZ gene of Escherichia coli and introduced the tycA-lacZ operon fusion by means of specialized transduction into sporulation mutants known to be blocked in sporulation-associated antibiotic production. Our principal finding was that tycA-directed lacZ expression was impaired in the stage-0 mutants with mutations spo0A, spo0B, and spo0E but not in spo0C, spo0F, spo0H, or spo+ bacteria. The dependence on the spo0A gene product could be entirely bypassed by an abrB suppressor mutation, which caused tycA-lacZ to be transcribed constitutively at all stages of growth. A simple model is proposed for the mechanism of tycA induction based on the Spo0A-dependent inactivation of Ab-B protein, which is proposed to be a negative regulator of tycA transcription.

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.

Pantetheine-Linked Peptide Intermediates in Gramicidin S and Tyrocidine Biosynthesis

To study the function of pantetheine in gramicidin S and tyrocidine biosynthesis, pepsin digests of the polymerizing enzymes, of which only the heavy ones contain pantetheine, were analyzed. The digests of gramicidin S enzymes charged with either [(14)C]proline or with D-phenylalanyl-[(14)C]proline, were analyzed by thin-layer chromatography; only the dipeptide showed a derivative associated with pantetheine. Similar results were obtained from the heavy tyrocidine enzyme charged with either [(14)C]asparagine alone or with the pentapeptide D-Phe-Pro-Phe-D-Phe-[(14)C]Asn. Several radioactive products appeared on the thin-layer chromatograms of both these digests; association with pantetheine was found only in the case of the pentapeptide. Exposure of the chromatogram from the pentapeptide-labeled digest to performic acid and development in a second direction separated the peptide from pantetheine, indicating that a nascent peptide was originally linked to the cofactor by a thioester bond. The connection of pantetheine only with peptide residues appears to confirm its role in transpeptidation during peptide chain growth.

Biosynthesis of tyrocidine by a cell-free enzyme system of Bacillus brevis ATCC 8185. IV. Further separation of component II into two fractions.

Journal Article Biosynthesis of Tyrocidine by a Cell-free Enzyme System of Bacillus brevis ATCC 8185: IV. Further Separation of Component II into Two Fractions Get access Michiyo KAMBE, Michiyo KAMBE The Institute for Protein Research, Osaka UniversityOsaka Search for other works by this author on: Oxford Academic PubMed Google Scholar Yohko SAKAMOTO, Yohko SAKAMOTO The Institute for Protein Research, Osaka UniversityOsaka Search for other works by this author on: Oxford Academic PubMed Google Scholar Kiyoshi KURAHASHI Kiyoshi KURAHASHI The Institute for Protein Research, Osaka UniversityOsaka Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 69, Issue 6, June 1971, Pages 1131–1133, https://doi.org/10.1093/oxfordjournals.jbchem.a129567 Published: 01 June 1971 Article history Received: 21 December 1970 Published: 01 June 1971

Biosynthesis of tyrocidine by a cell-free enzyme system of Bacillus brevis ATCC 8185. 3. Further purification of components I and II and their functions in tyrocidine synthesis.

Biosynthesis of tyrocidine by a cell-free enzyme system of Bacillus brevis ATCC 8185. 3. Further purification of components I and II and their functions in tyrocidine synthesis.

Isolation of enzyme-bound peptide intermediates in tyrocidine biosynthesis.

Isolation of enzyme-bound peptide intermediates in tyrocidine biosynthesis.

Tyrocidine biosynthesis by three complementary fractions from Bacillus brevis (ATCC 8185)

Tyrocidine biosynthesis by three complementary fractions from Bacillus brevis (ATCC 8185)

Enzyme-bound phosphopantetheine in tyrocidine biosynthesis

Phosphopantetheine has been demonstrated to be an enzyme-bound cofactor in one of the three enzyme fractions (light, intermediate, and heavy) required for tyrocidine biosynthesis. The enzyme-bound pantothenate had to be liberated by heating with alkali and treating the product with alkaline phosphatase. One mole of cofactor was estimated to be present per mole of heavy enzyme. It was absent from the light and intermediate enzyme fractions.

Biosynthesis of tyrocidine by A cell-free enzyme system of Bacillus brevis ATCC 8185 II. Amino acid substitution in tyrocidine

1. 1. The partially purified tyrocidine-synthesizing system of Bacillus brevis ATCC 8185 was revealed to have the ability to synthesize tyrocidines A, B, C and D. 2. 2. The enzyme system is not absolutely specific for particular amino acids. A new tyrocidine, tyrocidine E (tyrosine residue in tyrocidine A was replaced by phenylalanine), lysyl-tyrocidine A (ornithine residue in tyrocidine A was replaced by lysine) and isoleucyl-tyrocidine A (leucine residue in tyrocidine A was replaced by isoleucine) were synthesized in vitro. 3. 3. A single enzyme system catalyzed the synthesis of these tyrocidines and their formation was directly influenced by the amino acid concentrations in the reaction mixture. 4. 4. Some amino acid analogues, such as 5-methyltryptophan, p-fluorotryptophan, thienylalanine and p-fluorophenylalanine promoted the ATP- 32PP 1 exchange reaction and were incorporated into tyrocidine.

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.

FURTHER ASPECTS OF GRAMICIDIN AND TYROCIDINE BIOSYNTHESIS IN THE CELL-FREE SYSTEM OF BACILLUS BREVIS.

FURTHER ASPECTS OF GRAMICIDIN AND TYROCIDINE BIOSYNTHESIS IN THE CELL-FREE SYSTEM OF BACILLUS BREVIS.