The Biosynthesis of Gramicidin S in a Cell-Free System

Abstract

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of (14)C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [(14)C]-leucine, -proline and -phenylalanine over a period of 4hr. With [(14)C]leucine, incorporation into gramicidin S takes place in the range pH6-9 with maximum incorporation at pH7.0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [(14)C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.

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

“…The pH optimum (8.2-8.4) found previously for valine (Spaeren et al 1967) in a glycine buffer is in acceptable agreement with the present value (8.0-8.2). The value of 7 for the pH optimum for leucine in a different incubation mixture reported by Berg et al (1965) is in reasonable agreement with the present result.…”

“…A cell-free system prepared from Bacillus brevis that incorporates the five constituent amino acids in labelled form into gramicidin S in a different way to incorporation into protein was first described by Berg, Fr0holm & Laland (1965).…”

Incorporation of proline into prodigiosin by a Put mutant of Serratia marcesens

“…The difficulty to obtain a cell-free system may be due to the close association of prodigiosin biosynthesis with primary metabolism (16,18) and with enzymatic reactions that occur in the cell envelope (6,9). Similar problems have been encountered in cell-free studies on the biosynthesis of bacitracin (4, 7) and gramicidin S (1,20), two peptide antibiotics that incorporate certain amino acids into their structure. The syntheses of these two antibiotics have been studied successfully only recently in complex in vitro systems.…”

The chemical biology of modular biosynthetic enzymes

“…31 Surprisingly, the discovery of covalent channeling of intermediates in fatty acid biosynthesis also had an impact on the biosynthetic hypotheses for a family of cyclic peptides seemingly structurally unrelated to the acetate-based polyketides and fatty acids. 20 Christened ''nonribosomal peptides'' due to the insensitivity of their production to known inhibitors of ribosomal protein synthesis, 32,33 very little was understood about the biosynthetic origins of these compounds before 1968, when a series of seminal studies were performed in the laboratory of Lipmann at the Rockefeller University. Using activity-based fractionation, Lipmann and co-workers isolated crude preparations of the enzymes responsible for the production of the cyclic peptide antibiotic gramicidin S. By administration of radiolabeled amino acids and chemical cleavage studies it was quickly discovered that these enzymes utilized covalent aminoacyl-thioester intermediates as intermediates in the formation of nonribosomal peptides.…”