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The leader peptide of mutacin 1140 has distinct structural components compared to related class I lantibiotics.

Escano J, Stauffer B, Brennan J, Bullock M, Smith L - Microbiologyopen (2014)

Bottom Line: Mutacin 1140 leader peptide is structurally unique compared to other class I lantibiotic leader peptides.We have also determined that mutacin 1140 leader peptide contains a novel four amino acid motif compared to related lantibiotics.Our study on mutacin 1140 leader peptide provides a basis for future studies aimed at understanding its interaction with the PTM enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Texas A&M University, College Station, Texas, 77843.

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Identification of structural elements within the mutacin 1140 leader peptide that are important for bioactivity. (A) Covalent structure representation of the mutations made on the leader peptide. Bioactivity for leader peptide mutants were measured as the percent difference in the zone of inhibition between wild-type and the mutant strains. ΔlanA strain was used as a negative control for bioactivity in all experiments. The change in activity was measured for: (B) N-terminal deletions of the leader peptide, (C) mutations in the proposed FNLD-type box, (D) mutation in a new box, For each mutation, the bioactivity has been compared to the activity of wild-type S. mutans JH1140 strain. Statistical method used was Student t-test and the asterisk signifies statistical significance (P < 0.05).
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fig02: Identification of structural elements within the mutacin 1140 leader peptide that are important for bioactivity. (A) Covalent structure representation of the mutations made on the leader peptide. Bioactivity for leader peptide mutants were measured as the percent difference in the zone of inhibition between wild-type and the mutant strains. ΔlanA strain was used as a negative control for bioactivity in all experiments. The change in activity was measured for: (B) N-terminal deletions of the leader peptide, (C) mutations in the proposed FNLD-type box, (D) mutation in a new box, For each mutation, the bioactivity has been compared to the activity of wild-type S. mutans JH1140 strain. Statistical method used was Student t-test and the asterisk signifies statistical significance (P < 0.05).

Mentions: The mutacin 1140 leader peptide (Fig. 1B) is 18 and 11 amino acids longer than nisin A and epidermin leader peptides, respectively. Small consecutive truncations of four and five amino acids were made starting at the (-40) position corresponding to the amino acid after N-terminal methionine (Fig. 2A). Deletion mutations between residues Δ(-40 to -37), Δ(-36 to -33), Δ(-32 to -28) were investigated to determine whether there are regions of structural importance in the extended leader peptide (Fig. 2A). These mutations had little impact on the bioactivity of the mutant strains as determined by deferred antagonism assays (Fig. 2B). This assay is a sensitive quantitative measurement of bioactivity for mutacin 1140 production (Chen et al. 2013). Each strain of S. mutans is grown under identical conditions and the bioactivity is assessed by calculating the percent differences in the area of the zone of inhibition between mutant strains and wild-type strain. Reductions in activity suggest that less of mutacin 1140 is made or the biosynthesis of mutacin 1140 by the bacterium is altered leading to the synthesis of less active products. Progressively longer truncations from residues Δ(-40 to -33) and Δ(-40 to -28) were subsequently measured for bioactivity (Fig. 2A and B). The deletion mutants were reduced in bioactivity and the loss in bioactivity increased with the length of the deletion. The progressive loss in bioactivity with increasing size of deletion suggests that the length of the leader peptide is important for the biosynthesis of mutacin 1140. Mutacin 1140 was isolated from the Δ(-40 to -33) deletion mutant and its mass was 2265 Da, as predicted for a core peptide that has successfully undergone all dehydrations and decarboxylation (Table 3). There was no cyanylation of free thiols by CDAP, suggesting that all lanthionine rings were formed by the cyclase (Fig. 3). The substitution of six residues (-40 to -35) with histidines resulted in a statistically significant decrease in bioactivity, whereas, an insertion of six histidines between residues (-41 and -40) position resulted in no significant loss in bioactivity (Fig. 2B). These results further emphasize the importance of the leader peptide's predicted secondary structure for bioactivity (Fig. 1C). Secondary structure analysis using SOPMA (Geourjon and Deleage 1995) predicts that the N-terminal end of the leader peptide is an alpha helix, while the C-terminal end is a random coil (Fig. 1C). The insertion of six histidines at the N-terminal end did not affect bioactivity because they presumably extend outside of a binding cleft, whereas the substitution of six histidines is within the leader peptide-binding cleft of a PTM enzyme. The substitution of six histidines within this region is predicted by SOPMA to change the secondary structure to a random coil. Furthermore, the substitution of six histidines within this region would contribute to steric interference of binding. These results suggest that length of the first half of the leader peptide sequence, and possibly secondary structure, is more important for the biosynthesis of mutacin 1140 than the actual amino acid sequence. The lack of sequence specificity for the leader peptide has also been reported in other lantibiotic systems (van der Meer et al. 1994; Neis et al. 1997; Plat et al. 2011, 2013).


The leader peptide of mutacin 1140 has distinct structural components compared to related class I lantibiotics.

Escano J, Stauffer B, Brennan J, Bullock M, Smith L - Microbiologyopen (2014)

Identification of structural elements within the mutacin 1140 leader peptide that are important for bioactivity. (A) Covalent structure representation of the mutations made on the leader peptide. Bioactivity for leader peptide mutants were measured as the percent difference in the zone of inhibition between wild-type and the mutant strains. ΔlanA strain was used as a negative control for bioactivity in all experiments. The change in activity was measured for: (B) N-terminal deletions of the leader peptide, (C) mutations in the proposed FNLD-type box, (D) mutation in a new box, For each mutation, the bioactivity has been compared to the activity of wild-type S. mutans JH1140 strain. Statistical method used was Student t-test and the asterisk signifies statistical significance (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263518&req=5

fig02: Identification of structural elements within the mutacin 1140 leader peptide that are important for bioactivity. (A) Covalent structure representation of the mutations made on the leader peptide. Bioactivity for leader peptide mutants were measured as the percent difference in the zone of inhibition between wild-type and the mutant strains. ΔlanA strain was used as a negative control for bioactivity in all experiments. The change in activity was measured for: (B) N-terminal deletions of the leader peptide, (C) mutations in the proposed FNLD-type box, (D) mutation in a new box, For each mutation, the bioactivity has been compared to the activity of wild-type S. mutans JH1140 strain. Statistical method used was Student t-test and the asterisk signifies statistical significance (P < 0.05).
Mentions: The mutacin 1140 leader peptide (Fig. 1B) is 18 and 11 amino acids longer than nisin A and epidermin leader peptides, respectively. Small consecutive truncations of four and five amino acids were made starting at the (-40) position corresponding to the amino acid after N-terminal methionine (Fig. 2A). Deletion mutations between residues Δ(-40 to -37), Δ(-36 to -33), Δ(-32 to -28) were investigated to determine whether there are regions of structural importance in the extended leader peptide (Fig. 2A). These mutations had little impact on the bioactivity of the mutant strains as determined by deferred antagonism assays (Fig. 2B). This assay is a sensitive quantitative measurement of bioactivity for mutacin 1140 production (Chen et al. 2013). Each strain of S. mutans is grown under identical conditions and the bioactivity is assessed by calculating the percent differences in the area of the zone of inhibition between mutant strains and wild-type strain. Reductions in activity suggest that less of mutacin 1140 is made or the biosynthesis of mutacin 1140 by the bacterium is altered leading to the synthesis of less active products. Progressively longer truncations from residues Δ(-40 to -33) and Δ(-40 to -28) were subsequently measured for bioactivity (Fig. 2A and B). The deletion mutants were reduced in bioactivity and the loss in bioactivity increased with the length of the deletion. The progressive loss in bioactivity with increasing size of deletion suggests that the length of the leader peptide is important for the biosynthesis of mutacin 1140. Mutacin 1140 was isolated from the Δ(-40 to -33) deletion mutant and its mass was 2265 Da, as predicted for a core peptide that has successfully undergone all dehydrations and decarboxylation (Table 3). There was no cyanylation of free thiols by CDAP, suggesting that all lanthionine rings were formed by the cyclase (Fig. 3). The substitution of six residues (-40 to -35) with histidines resulted in a statistically significant decrease in bioactivity, whereas, an insertion of six histidines between residues (-41 and -40) position resulted in no significant loss in bioactivity (Fig. 2B). These results further emphasize the importance of the leader peptide's predicted secondary structure for bioactivity (Fig. 1C). Secondary structure analysis using SOPMA (Geourjon and Deleage 1995) predicts that the N-terminal end of the leader peptide is an alpha helix, while the C-terminal end is a random coil (Fig. 1C). The insertion of six histidines at the N-terminal end did not affect bioactivity because they presumably extend outside of a binding cleft, whereas the substitution of six histidines is within the leader peptide-binding cleft of a PTM enzyme. The substitution of six histidines within this region is predicted by SOPMA to change the secondary structure to a random coil. Furthermore, the substitution of six histidines within this region would contribute to steric interference of binding. These results suggest that length of the first half of the leader peptide sequence, and possibly secondary structure, is more important for the biosynthesis of mutacin 1140 than the actual amino acid sequence. The lack of sequence specificity for the leader peptide has also been reported in other lantibiotic systems (van der Meer et al. 1994; Neis et al. 1997; Plat et al. 2011, 2013).

Bottom Line: Mutacin 1140 leader peptide is structurally unique compared to other class I lantibiotic leader peptides.We have also determined that mutacin 1140 leader peptide contains a novel four amino acid motif compared to related lantibiotics.Our study on mutacin 1140 leader peptide provides a basis for future studies aimed at understanding its interaction with the PTM enzymes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Texas A&M University, College Station, Texas, 77843.

Show MeSH
Related in: MedlinePlus