Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Feb;62(2):385–392. doi: 10.1128/aem.62.2.385-392.1996

Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5.

G Bierbaum 1, C Szekat 1, M Josten 1, C Heidrich 1, C Kempter 1, G Jung 1, H G Sahl 1
PMCID: PMC167809  PMID: 8593044

Abstract

Pep5 is a 34-amino-acid antimicrobial peptide, produced by Staphylococcus epidermidis 5, that contains the thioether amino acids lanthionine and methyllanthionine, which form three intramolecular ring structures. In addition, two didehydrobutyrines are present in the central part of the lantibiotic and an oxobutyryl residue is located at the N terminus. All rare amino acids are introduced by posttranslational modifications of a ribosomally made precursor peptide. To elucidate the function of the modified residues for the antimicrobial action of Pep5, mutant peptides, in which single modified residues had been eliminated, were produced by site-directed mutagenesis. All of these peptides showed a reduced antimicrobial activity. In addition, those peptides from which the ring structures had been deleted became susceptible to proteolytic digest. This demonstrates that the ring structures serve as stabilizers of conformations essential for activity, e.g., amphiphilicity, as well as for protecting Pep5 against proteases of the producing strains. In addition, residues that could serve as precursors of new modified amino acids in lantibiotics were introduced into the Pep5 precursor peptide. This way, a novel methyllanthionine and a didehydroalanine were inserted into the flexible central part of Pep5, demonstrating that biosynthesis of modified amino acids is feasible by protein engineering and use of the lantibiotic modification system.

Full Text

The Full Text of this article is available as a PDF (303.0 KB).

Selected References

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

  1. Augustin J., Götz F. Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation. FEMS Microbiol Lett. 1990 Jan 1;54(1-3):203–207. doi: 10.1016/0378-1097(90)90283-v. [DOI] [PubMed] [Google Scholar]
  2. Augustin J., Rosenstein R., Wieland B., Schneider U., Schnell N., Engelke G., Entian K. D., Götz F. Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur J Biochem. 1992 Mar 15;204(3):1149–1154. doi: 10.1111/j.1432-1033.1992.tb16740.x. [DOI] [PubMed] [Google Scholar]
  3. Bierbaum G., Reis M., Szekat C., Sahl H. G. Construction of an expression system for engineering of the lantibiotic Pep5. Appl Environ Microbiol. 1994 Dec;60(12):4332–4338. doi: 10.1128/aem.60.12.4332-4338.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bierbaum G., Sahl H. G. Autolytic system of Staphylococcus simulans 22: influence of cationic peptides on activity of N-acetylmuramoyl-L-alanine amidase. J Bacteriol. 1987 Dec;169(12):5452–5458. doi: 10.1128/jb.169.12.5452-5458.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bisschop A., Konings W. N. Reconstitution of reduced nicotinamide adenine dinucleotide oxidase activity with menadione in membrane vesicles from the menaquinone-deficient Bacillus subtilis aro D. Relation between electron transfer and active transport. Eur J Biochem. 1976 Aug 16;67(2):357–365. doi: 10.1111/j.1432-1033.1976.tb10699.x. [DOI] [PubMed] [Google Scholar]
  6. Driessen A. J., van den Hooven H. W., Kuiper W., van de Kamp M., Sahl H. G., Konings R. N., Konings W. N. Mechanistic studies of lantibiotic-induced permeabilization of phospholipid vesicles. Biochemistry. 1995 Feb 7;34(5):1606–1614. doi: 10.1021/bi00005a017. [DOI] [PubMed] [Google Scholar]
  7. Ersfeld-Dressen H., Sahl H. G., Brandis H. Plasmid involvement in production of and immunity to the staphylococcin-like peptide Pep 5. J Gen Microbiol. 1984 Nov;130(11):3029–3035. doi: 10.1099/00221287-130-11-3029. [DOI] [PubMed] [Google Scholar]
  8. Freund S., Jung G., Gutbrod O., Folkers G., Gibbons W. A., Allgaier H., Werner R. The solution structure of the lantibiotic gallidermin. Biopolymers. 1991 May;31(6):803–811. doi: 10.1002/bip.360310626. [DOI] [PubMed] [Google Scholar]
  9. Gross E., Morell J. L. The structure of nisin. J Am Chem Soc. 1971 Sep 8;93(18):4634–4635. doi: 10.1021/ja00747a073. [DOI] [PubMed] [Google Scholar]
  10. Kaletta C., Entian K. D., Kellner R., Jung G., Reis M., Sahl H. G. Pep5, a new lantibiotic: structural gene isolation and prepeptide sequence. Arch Microbiol. 1989;152(1):16–19. doi: 10.1007/BF00447005. [DOI] [PubMed] [Google Scholar]
  11. Kordel M., Benz R., Sahl H. G. Mode of action of the staphylococcinlike peptide Pep 5: voltage-dependent depolarization of bacterial and artificial membranes. J Bacteriol. 1988 Jan;170(1):84–88. doi: 10.1128/jb.170.1.84-88.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kuipers O. P., Rollema H. S., Yap W. M., Boot H. J., Siezen R. J., de Vos W. M. Engineering dehydrated amino acid residues in the antimicrobial peptide nisin. J Biol Chem. 1992 Dec 5;267(34):24340–24346. [PubMed] [Google Scholar]
  13. Kupke T., Kempter C., Jung G., Götz F. Oxidative decarboxylation of peptides catalyzed by flavoprotein EpiD. Determination of substrate specificity using peptide libraries and neutral loss mass spectrometry. J Biol Chem. 1995 May 12;270(19):11282–11289. doi: 10.1074/jbc.270.19.11282. [DOI] [PubMed] [Google Scholar]
  14. Kupke T., Stevanović S., Sahl H. G., Götz F. Purification and characterization of EpiD, a flavoprotein involved in the biosynthesis of the lantibiotic epidermin. J Bacteriol. 1992 Aug;174(16):5354–5361. doi: 10.1128/jb.174.16.5354-5361.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Liu W., Hansen J. N. The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms. Appl Environ Microbiol. 1993 Feb;59(2):648–651. doi: 10.1128/aem.59.2.648-651.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Maloy W. L., Kari U. P. Structure-activity studies on magainins and other host defense peptides. Biopolymers. 1995;37(2):105–122. doi: 10.1002/bip.360370206. [DOI] [PubMed] [Google Scholar]
  17. Meyer C., Bierbaum G., Heidrich C., Reis M., Süling J., Iglesias-Wind M. I., Kempter C., Molitor E., Sahl H. G. Nucleotide sequence of the lantibiotic Pep5 biosynthetic gene cluster and functional analysis of PepP and PepC. Evidence for a role of PepC in thioether formation. Eur J Biochem. 1995 Sep 1;232(2):478–489. doi: 10.1111/j.1432-1033.1995.tb20834.x. [DOI] [PubMed] [Google Scholar]
  18. Ojcius D. M., Young J. D. Cytolytic pore-forming proteins and peptides: is there a common structural motif? Trends Biochem Sci. 1991 Jun;16(6):225–229. doi: 10.1016/0968-0004(91)90090-i. [DOI] [PubMed] [Google Scholar]
  19. Ottenwälder B., Kupke T., Brecht S., Gnau V., Metzger J., Jung G., Götz F. Isolation and characterization of genetically engineered gallidermin and epidermin analogs. Appl Environ Microbiol. 1995 Nov;61(11):3894–3903. doi: 10.1128/aem.61.11.3894-3903.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Reis M., Eschbach-Bludau M., Iglesias-Wind M. I., Kupke T., Sahl H. G. Producer immunity towards the lantibiotic Pep5: identification of the immunity gene pepI and localization and functional analysis of its gene product. Appl Environ Microbiol. 1994 Aug;60(8):2876–2883. doi: 10.1128/aem.60.8.2876-2883.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sahl H. G., Brandis H. Production, purification and chemical properties of an antistaphylococcal agent produced by Staphylococcus epidermidis. J Gen Microbiol. 1981 Dec;127(2):377–384. doi: 10.1099/00221287-127-2-377. [DOI] [PubMed] [Google Scholar]
  22. Sahl H. G., Grossgarten M., Widger W. R., Cramer W. A., Brandis H. Structural similarities of the staphylococcin-like peptide Pep-5 to the peptide antibiotic nisin. Antimicrob Agents Chemother. 1985 May;27(5):836–840. doi: 10.1128/aac.27.5.836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sahl H. G., Jack R. W., Bierbaum G. Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. Eur J Biochem. 1995 Jun 15;230(3):827–853. doi: 10.1111/j.1432-1033.1995.tb20627.x. [DOI] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schnell N., Entian K. D., Schneider U., Götz F., Zähner H., Kellner R., Jung G. Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature. 1988 May 19;333(6170):276–278. doi: 10.1038/333276a0. [DOI] [PubMed] [Google Scholar]
  26. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  27. Vogel H., Nilsson L., Rigler R., Meder S., Boheim G., Beck W., Kurth H. H., Jung G. Structural fluctuations between two conformational states of a transmembrane helical peptide are related to its channel-forming properties in planar lipid membranes. Eur J Biochem. 1993 Mar 1;212(2):305–313. doi: 10.1111/j.1432-1033.1993.tb17663.x. [DOI] [PubMed] [Google Scholar]
  28. Wade D., Andreu D., Mitchell S. A., Silveira A. M., Boman A., Boman H. G., Merrifield R. B. Antibacterial peptides designed as analogs or hybrids of cecropins and melittin. Int J Pept Protein Res. 1992 Nov;40(5):429–436. doi: 10.1111/j.1399-3011.1992.tb00321.x. [DOI] [PubMed] [Google Scholar]
  29. Weil H. P., Beck-Sickinger A. G., Metzger J., Stevanovic S., Jung G., Josten M., Sahl H. G. Biosynthesis of the lantibiotic Pep5. Isolation and characterization of a prepeptide containing dehydroamino acids. Eur J Biochem. 1990 Nov 26;194(1):217–223. doi: 10.1111/j.1432-1033.1990.tb19446.x. [DOI] [PubMed] [Google Scholar]
  30. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  31. Zell R., Fritz H. J. DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues. EMBO J. 1987 Jun;6(6):1809–1815. doi: 10.1002/j.1460-2075.1987.tb02435.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES