Gallidermin: a new lanthionine-containing polypeptide antibiotic

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The antibacterial peptide SA-FF22, produced by the pathogen Streptococcus pyogenes strain FF22 was purified and features of its primary and secondary structure were characterised. Mass spectrometry demonstrated the pure peptide had a mass of 2794Da while, amino acid analysis revealed the presence of the unusual, thioether amino acids lanthionine and 3-methyllanthionine ; thus SA-FF22 is a member of the group of antibacterial polypeptides termed lantibiotics. Furthermore, amino acid sequencing showed a unique sequence which was blocked at position 23 by a residue of the unsaturated amino acid 2,3-didehydrobutyrine. Carboxypeptidase-Y digestion could be used to demonstrate that serine occupies the C-terminal position only after complete oxidation of the thioether amino acid bridges, suggesting that the three-dimensional structure of the native peptide may prevent access of the enzyme to the C-terminus. Fragmentation of the native peptide with a variety of proteolytic enzymes failed to yield a peptide containing less than all three of the cross-linked lanthionine and methyllanthionine residues and demonstrated that all three thioether bridges overlapped. Analysis of the circular dichroism of SA-FF22 in various concentrations of 2,2,2-trifluoroethanol in water, SDS micelles and in the presence of artificial phospholipid vesicles suggested that there is significant change in its secondary structure from aqueous to lipophilic environments.SA-FF22 is a bacteriocin-like inhibitory substance produced by Streptococcus pyogenes strain FF22 and which has been shown to be bactericidal for many strains of related streptococci [l]. Subsequent to its discovery, SA-FF22 was partially purified and characterised [2, 31. In these initial studies it was demonstrated that the inhibitory factor contained an essential proteinaceous component since it was inactivated by several proteolytic enzymes, was of approximately 8000Da as determined by dialysis and gel filtration, was stable in acid solution and at high temperatures but was not stable under alkaline conditions and was active against only certain Gram-positive bacteria. Further studies showed that the genetic determinant(s) that controlled SA-FF22 production appeared to be lost spontaneously in aged cultures or through the use of plasmid 'curing' agents, suggesting that SA-FF22 production was encoded on a plasmid. AdditionCorrespondence to R. W. Jack,

Section: Further Enzyme Digestionssupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Elucidation of the structure of SA‐FF22, a lanthionine‐containing antibacterial peptide produced by Streptococcus pyogenes strain FF22

Jack

Carne

Metzger

et al. 1994

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“…Naturally occurring variants are not uncommon within the family of lantibiotics ; they include gallidermin and epidermin, produced by different Staphylococcus strains, that differ only in a Leu/Ile substitution [14], and cinnamycin and duramycin, both produced by Streptomyces cinnamorieus, that differ by a Arg/Lys substitution [15].…”

Section: Nucleotide and Deduced Amino Acid Sequence Of The Nisz Genementioning

confidence: 99%

Identification and characterization of the lantibiotic nisin Z, a natural nisin variant

Mulders

Boerrigter

Rollema

et al. 1991

390

234

Lactococcus lactis strain NIZO 221 86 produces an extracellular, lanthionine-containing 3.5-kDa polypeptide with antimicrobial activity. Its retention time on reversed-phase (RP) HPLC and its amino acid composition showed high similarities but no complete identity to nisin. The gene for this lantibiotic, designated nisZ, has been cloned and its nucleotide sequence was found to be identical to that of the precursor nisin gene apart from a single mutation resulting in the substitution His27Asn in the mature polypeptide. NMR studies of the natural nisin variant, which has been designated nisin Z, confirmed the His27Asn substitution and indicated that it has a similar structure to nisin.Lantibiotics are ribosomally synthesized antimicrobial polypeptides containing the unusual amino acids lanthionine and fl-methyllanthionine [I]. Genetic analysis has shown that the lantibiotics are produced as precursor molecules consisting of an uncommon leader region and a structural region that is post-translationally processed [1 -51. Nisin is a 34-aminoacid lantibiotic produced by Lactococcus lactis that has wide applications as a food preservative 161. It also contains dehydroalanine and dehydrobutyrine (P-methyldehydroalanine) [7]. An extensive characterization of nisin by onedimensional and two-dimensional NMR analysis has recently been described [8, 91. Long range contacts were observed in the NOE spectra of nisin, indicating the presence of a preferred conformation [8]. In order to obtain further insight into the structure/function relationship of nisin, we purified and characterized an antimicrobial peptide produced by L. luctis strain NIZO 22186 that showed high similarity to nisin. Our detailed analysis on the biochemical and genetic level shows that this compound is a natural nisin variant, designated nisin Z, that differs from nisin in a single amino acid. MATERIALS AND METHODS Cultivation and purification L . luctis, subspecies luctis, strains NIZO R5 [lo] and NIZO22186 (NIZO collection) were grown for 16 h at 30°C in a medium containing 1% sucrose, 1% peptone, 1% yeast extract, 0.2% NaCI, 0.002% MgS04 . 7 H 2 0 and 1 YO KH2P04, pH 6.8 (L. de Vuyst, personal communication). After addition of 0.5 M (NH4)2S04, the cell-free supernatant (5 1) was run on a Fractogel TSK butyl650-S (Merck) column (5 x 20 cm), Correspondence to W. M. de Vos, Netherlands Institute for DairyAbbreviations. RP, reversed phase; Ile, isoleucine. Note. The novel nucleotide sequence data published here has been deposited with the EMBL sequence data banks and is available under accession number X61144.The novel amino acid sequence data published here has been deposited with the EMBL sequence data bank.Research (NIZO), P.O. Box 20, NL-6710 BA Ede, The Netherlands which was washed with 1.51 0.5M (NH4)2S04 and subsequently with deionized water until A220 < 0.5 was achieved. The antimicrobial substance was then eluted using 10mM HC1 and concentrated by ultrafiltration (Filtron, Novacell, Omega, molecular mass cut off 1 kDa). Final purification and analy...

“…The prelantibiotics described until now [I, 20 -261 belonged to the group of cationic and membrane-modifying lantibiotics epidermin [32-341, nisin [35], subtilin [36], Pep5 [37] and gallidermin [38]. Their structural genes [I, 20-261 show a common organization and general similarities.…”

Section: Comparison Of the Preluntibiotic Of Ro 09-0198 With Other Rementioning

confidence: 99%

Prepeptide sequence of cinnamycin (Ro 09–0198): the first structural gene of a duramycin‐type lantibiotic

Kaletta

Entian

Jung

1991

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The tetracyclic polypeptide antibiotic cinnamycin (Ro 90-0198) belongs to the duramycin-type lantibiotics and contains the unusual amino acids threo-3-methyl-lanthionine, meso-lanthionine, lysinoalanine and 3-hydroxyaspartic acid. Its structural gene, referred to as cinA, has been identified on isolated chromosomal DNA of the Ro 09-01 98-producing strain Streptoverticillium griseoverticillutum via a 39-residue oligonucleotide probe derived from fragment 7 -19 of the hypothetical prolantibiotic sequence CRQSCSFGPFTFVCDGNTK. This propeptide part was then found within an open reading frame of 77 amino acids. In contrast to the nisin-type prelantibiotics, this first duramycin-type prelantibiotic has an unusually long leader sequence of 58 amino acids. It also differs in the processing site and the direction of the formation of the threo-3-methyl-lanthionine bridges is from N-terminal cysteine to C-terminal dehydrated threonine residues, whereas the meso-lanthionine and lysinoalanine bridges are formed by addition reactions from C-terminal cysteine or lysine to N-terminal dehyrated serine residues.