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Studies on the biosynthesis of gramicidin S in a cell-free system from Bacillus brevis. Further attempts to elucidate its mechanism of synthesis.

ABSTRACT: 1. The pH optima for the incorporation of (14)C-labelled amino acids into gramicidin S by an 11000g cell-free extract from Bacillus brevis have been determined. The pH optima for leucine, proline, phenylalanine, ornithine and valine were 7.5-7.7, 7.5-7.7, 7.7-7.9, 7.7-7.9 and 8.0-8.2 respectively. Hence the greatest difference in pH optima existed between leucine and valine, where it was 0.5pH unit. 2. The 11000g cell-free extract incorporated into gramicidin S only the l-isomers of valine, proline and ornithine. However, both isomers of leucine are utilized and the experiments indicate that a leucine racemase exists in the 11000g cell-free extract. With phenylalanine the l-isomer is utilized much more effectively than the d-isomer. This is noteworthy since it is the d-isomer that occurs in gramicidin S. The experiments indicate that conversion of the l-isomer into the d-form takes place at a stage beyond that of the free amino acid.

SUBMITTER: Figenschou KJ

PROVIDER: S-EPMC1198330 | biostudies-other | 1967 Nov

REPOSITORIES: biostudies-other

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Project description: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.

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Studies on the biosynthesis of gramicidin S in a cell-free system from Bacillus brevis. Further attempts to elucidate its mechanism of synthesis.

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