Dissecting Structural and Functional Diversity of the Lantibiotic Mersacidin

Abstract

SummaryMersacidin is a tetracyclic lantibiotic with antibacterial activity against Gram-positive pathogens. To probe the specificity of the biosynthetic pathway of mersacidin and obtain analogs with improved antibacterial activity, an efficient system for generating variants of this lantibiotic was developed. A saturation mutagenesis library of the residues of mersacidin not involved in cycle formation was constructed and used to validate this system. Mersacidin analogs were obtained in good yield in approximately 35% of the cases, producing a collection of 82 new compounds. This system was also used for the production of deletion and insertion mutants of mersacidin. The outcome of these studies suggests that this system can be extended to produce mersacidin variants with multiple changes that will allow a full investigation of the potential use of modified mersacidins as therapeutic agents.

Fundamental functionality: recent developments in understanding the structure–activity relationships of lantibiotic peptides

“…19 Very surprisingly, this mutation did not abolish activity, unlike with the equivalent mersacidin and haloduracin-a mutants that were both strongly affected by this change. 8,[20][21][22] Other mutants in ring A were found to retain activity, suggesting there is some degree of flexibility in the amino acids of this ring. 23 Mutagenesis has been used to produce weakly active analogs containing the changes S4T, H8P, Q20T and truncation at S27.…”

“…21 A subsequent systematic production of mersacidin analogs via mutagenesis yielded a far larger number of mutants. 22 Three libraries were designed in which amino acids were substituted, inserted or deleted. None of the shortened peptides (amino-acid deletions) were produced by this system, suggesting that contraction of the rings produces substrates that cannot be processed by the mersacidin biosynthetic enzymes.…”

“…This result leaves the importance of the melan ring unknown. 22 Although most of the mutations that retained some degree of activity were found in the second ring of mersacidin, those that showed the greatest activity were spread throughout the molecule. This suggests that although the ring B is a good place to focus further analog studies, it should by no means be to the exclusion of the rest of the molecule.…”

Investigating the importance of charged residues in lantibiotics

“…1 It was noted that five of the six variants in which charged residues were replaced with alanine (i.e., Ltnα D10A, Ltnα H23A, LtnαK30A, Ltnβ K24A and that of the wild-type peptide, the activity of both P6H and G8H was reduced. 34 As a consequence of the site-saturation mutagenesis of nukacin ISK-1, a large collection of charge-altered mutants are available. The majority of these have been assessed through bioactivity-based studies alone (i.e., studies in which the antimicrobial activity of individual lantibioticproducing strains rather than equimolar concentrations of purified peptides) and these have revealed that mutagenesis of the N-terminal lysine's, K1, K2 and K3, is generally well tolerated except when aspartate or glutamate residues are incorporated.…”

Organization of the biosynthetic genes encoding deoxyactagardine B (DAB), a new lantibiotic produced by Actinoplanes liguriae NCIMB41362

“…Together with the reported promising antibacterial activities of this class of compound, the lantibiotics now represent attractive precursors for generating variants with improved activity. 10,11 However, information regarding the organization of the biosynthetic genes of lantibiotics, particularly from G-C rich organisms, is still relatively limited. In this study, we have described the cloning and analysis of the biosynthetic gene cluster encoding deoxyactagardine B (DAB), a hitherto uncharacterized lantibiotic.…”