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. 1971 Sep;68(9):2069–2072. doi: 10.1073/pnas.68.9.2069

Pantetheine-Linked Peptide Intermediates in Gramicidin S and Tyrocidine Biosynthesis

Horst Kleinkauf 1,*, Robert Roskoski Jr 1, Fritz Lipmann 1,†
PMCID: PMC389353  PMID: 4943784

Abstract

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 [14C]proline or with D-phenylalanyl-[14C]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 [14C]asparagine alone or with the pentapeptide D-Phe-Pro-Phe-D-Phe-[14C]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.

Keywords: pepsin, thin-layer chromatography, thioesters, enzyme complex

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Frøshov Ø, Zimmer T. L., Laland S. G. The nature of the enzyme bound intermediates in gramicidin s biosynthesis. FEBS Lett. 1970 Mar 16;7(1):68–71. doi: 10.1016/0014-5793(70)80620-2. [DOI] [PubMed] [Google Scholar]
  2. Gevers W., Kleinkauf H., Lipmann F. Peptidyl transfers in gramicidin S bisoynthesis from enzyme-bound thioester intermediates. Proc Natl Acad Sci U S A. 1969 Aug;63(4):1335–1342. doi: 10.1073/pnas.63.4.1335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gevers W., Kleinkauf H., Lipmann F. The activation of amino acids for biosynthesis of gramicidin S. Proc Natl Acad Sci U S A. 1968 May;60(1):269–276. doi: 10.1073/pnas.60.1.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gilhuus-Moe C. C., Kristensen T., Bredesen J. E., Zimmer T. -L., Laland S. G. The presence and possible role of phosphopantothenic acid in gramicidin S synthetase. FEBS Lett. 1970 Apr 16;7(3):287–290. doi: 10.1016/0014-5793(70)80183-1. [DOI] [PubMed] [Google Scholar]
  5. Kleinkauf H., Gevers W., Lipmann F. Interrelation between activation and polymerization in gramicidin S biosynthesis. Proc Natl Acad Sci U S A. 1969 Jan;62(1):226–233. doi: 10.1073/pnas.62.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kleinkauf H., Gevers W. Nonribosomal polypeptide synthesis: the biosynthesis of a cyclic peptide antibiotic, gramicidin S. Cold Spring Harb Symp Quant Biol. 1969;34:805–813. doi: 10.1101/sqb.1969.034.01.092. [DOI] [PubMed] [Google Scholar]
  7. Kleinkauf H., Gevers W., Roskoski R., Jr, Lipmann F. Enzyme-bound phosphopantetheine in tyrocidine biosynthesis. Biochem Biophys Res Commun. 1970 Dec 9;41(5):1218–1222. doi: 10.1016/0006-291x(70)90216-0. [DOI] [PubMed] [Google Scholar]
  8. Pugh E. L., Wakil S. J. Studies on the mechanism of fatty acid synthesis. XIV. The prosthetic group of acyl carrier protein and the mode of its attachment to the protein. J Biol Chem. 1965 Dec;240(12):4727–4733. [PubMed] [Google Scholar]
  9. Roskoski R., Jr, Gevers W., Kleinkauf H., Lipmann F. Tyrocidine biosynthesis by three complementary fractions from Bacillus brevis (ATCC 8185). Biochemistry. 1970 Dec 8;9(25):4839–4845. doi: 10.1021/bi00827a002. [DOI] [PubMed] [Google Scholar]
  10. Roskoski R., Jr, Kleinkauf H., Gevers W., Lipmann F. Isolation of enzyme-bound peptide intermediates in tyrocidine biosynthesis. Biochemistry. 1970 Dec 8;9(25):4846–4851. doi: 10.1021/bi00827a003. [DOI] [PubMed] [Google Scholar]
  11. Roskoski R., Jr, Ryan G., Kleinkauf H., Gevers W., Lipmann F. Polypeptide biosynthesis form thioesters of amino acids. Arch Biochem Biophys. 1971 Apr;143(2):485–492. doi: 10.1016/0003-9861(71)90233-5. [DOI] [PubMed] [Google Scholar]
  12. Tomino S., Yamada M., Itoh H., Kurahashik Cell-free synthesis of gramicidin S. Biochemistry. 1967 Aug;6(8):2552–2560. doi: 10.1021/bi00860a037. [DOI] [PubMed] [Google Scholar]

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