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

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.

Organization of the genes encoding the biosynthesis of actagardine and engineering of a variant generation system

“…Alanine scanning was performed to further validate the engineered variant generation system and provide an understanding of the tolerance of the biosynthetic machinery to modifications of the amino acid sequence, Previous studies examining alterations to residues involved in bridge formation have predominantly resulted in either a complete loss of activity or a significant reduction in the level of activity of the resulting lantibiotic variant (Ottenwälder et al, 1995;Bierbaum et al, 1996;Cotter et al, 2006;Cooper et al, 2008). Given that the pattern of lanthionine bridges of actagardine is conserved among several lantibiotics, including mersacidin (Prasch et al, 1997), lacticin 3147-A2 (Ryan et al, 1999), plantaricin Wa (Holo et al, 2001), staphylococcin C55a (Navaratna et al, 1998) and haloduracin (McClerren et al, 2006), and the apparent significance of these bridges for conformational stability and retention of activity, we chose only to mutagenize residues not involved in bridge formation of the actagardine molecule.…”

The biology of lantibiotics from the lacticin 481 group is coming of age

“…In some cases, Ser11 or Ser18 is replaced by a threonine, the dehydration of which results in a MeLan instead of a Lan residue. biologically active conformation, thereby explaining at least in part their stability (Bierbaum et al, 1996b). In addition, (Me)Lan residues stabilize lantibiotics against proteases by restricting the conformational freedom of potential cleavage sites (Bierbaum et al, 1996b).…”

Identification of the biosynthesis gene cluster for the novel lantibiotic paenilan fromPaenibacillus polymyxaE681 and characterization of its product

“…This is because of the continuous discovery of novel lantibiotics with diverse structures and antimicrobial activity against important pathogenic strains (Cotter et al 2005). The low incidence of resistance (Montalban-Lopez et al 2011;Cotter et al 2013;Draper et al 2015) and high heat and protease stability arising from the intramolecular thioether cross links are also seen as benefits of lantibiotics that broaden their application potential in medicines, prophylactics and as additives in personal healthcare products and as food preservatives (Bierbaum et al 1996;Lubelski et al 2008).…”