Complete alanine scanning of the two‐component lantibiotic lacticin 3147: generating a blueprint for rational drug design

Abstract

SummaryLantibiotics are post-translationally modified antimicrobial peptides which are active at nanomolar concentrations. Some lantibiotics have been shown to function by targeting lipid II, the essential precursor of cell wall biosynthesis. Given that lantibiotics are ribosomally synthesized and amenable to sitedirected mutagenesis, they have the potential to serve as biological templates for the production of novel peptides with improved functionalities. However, if a rational approach to novel lantibiotic design is to be adopted, an appreciation of the roles of each individual amino acid (and each domain) is required. To date no lantibiotic has been subjected to such rigorous analysis. To address this issue we have carried out complete scanning mutagenesis of each of the 59 amino acids in lacticin 3147, a twocomponent lantibiotic which acts through the synergistic activity of the peptides LtnA1 (30 amino acids) and LtnA2 (29 amino acids). All mutations were performed in situ in the native 60kb plasmid, pMRC01. A number of mutations resulted in the elimination of detectable bioactivity and seem to represent an invariable core within these and related peptides. Significantly however, of the 59 amino acids, at least 36 can be changed without resulting in a complete loss of activity. Many of these are clustered to form variable domains within the peptides. The information generated in this study represents a blue-print that will be critical for the rational design of lantibioticbased antimicrobial compounds.

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

“…Of the other peptides produced, none were more active than the natural peptides, and overall, it was found that the A2 peptide (particularly it's N terminus) was more tolerant to change than A1. 40 A further mutagenesis study was undertaken to probe for mutants that decreased the activity of the natural peptides. More mutants were detected for A1 (as it is less flexible to amino-acid alteration) than A2.…”

“…On one hand, Ross, and coworkers have utilized a mutagenesis approach to produce a large number of single amino-acid mutants to examine the importance of each amino acid in the two-component peptide system. [39][40][41][42] On the other hand, Vederas and coworkers have used chemical synthesis to investigate the importance of the lan and melan rings with a view towards producing simpler and more stable analogs. [43][44][45] HALODURACIN Haloduracin is a two-component lantibiotic isolated from Bacillus halodurans consisting of a globular a-peptide resembling the class B lantibiotics and an elongated b-peptide similar to class A.…”

Investigating the importance of charged residues in lantibiotics

“…These peptides do however share one common charge-related feature in that all possess a conserved glutamate residue (referred to in the context of nukacin ISK-1 above) which is thought to play a key role in receptor binding, and mutagenesis of which resulted in the abolition of antimicrobial activity. 10,33,40,41 In contrast, β peptides are linear, appear to represent the pore-forming component and thus more closely resemble classical cationic antimicrobial peptides. Bliβ of licheniciden is the only β component which contains negatively charged residues and as a result has a neutral rather than a cationic charge.…”

“…The nisin peptides referred to are derivatives of two natural forms of nisin, i.e., nisin A and Z, which differ by one amino acid in that nisin A and Z have a histidine and globular do not resemble classical cationic antimicrobial peptides. Indeed two of these peptides, Ltnα of Lacticin 3147, 33,37 and putative Pnmα of Pneumococcin SP23-BS72, 14 have a net neutral charge. These peptides also resemble a number of one peptide lantibiotics such as the aforementioned mersacidin and actagardine which are anionic, 7,10,17,38,39 thereby establishing that an overall net positive charge is not an essential pre-requisite for this extended group of peptides.…”

Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine cyanobacteria

“…However, LanM enzymes reveal low substrate specificity under laboratory conditions, a property that has been exploited for the generation of variants of the natural products (12)(13)(14). Wondering whether organisms in nature have taken advantage of this substrate promiscuity to generate natural product libraries with a single streamlined biosynthetic toolset, we conducted a search of the sequenced bacterial genomes for cases where a single bacterium can produce multiple lanthionine-containing peptides using the same LanM enzyme.…”