Lantibiotics are ribosomally synthesized peptides that undergo posttranslational modifications to their mature, antimicrobial form. They are characterized by the unique amino acids lanthionine and methyllanthionine, introduced by means of dehydration of Ser͞Thr residues followed by reaction of the resulting dehydro amino acids with cysteines to form thioether linkages. Two-component lantibiotics use two peptides that are each posttranslationally modified to yield two functionally distinct products that act in synergy to provide bactericidal activity. By using genetic data instead of isolation, a two-component lantibiotic, haloduracin, was identified in the genome of the Gram-positive alkaliphilic bacterium Bacillus halodurans C-125. We show that heterologously expressed and purified precursor peptides HalA1 and HalA2 are processed by the purified modification enzymes HalM1 and HalM2 in an in vitro reconstitution of the biosynthesis of a two-component lantibiotic. The activity of each HalM enzyme is substratespecific, and the assay products exhibit antimicrobial activity after removal of their leader sequences at an engineered Factor Xa cleavage site, indicating that correct thioether formation has occurred. Haloduracin's biological activity depends on the presence of both modified peptides. The structures of the two mature haloduracin peptides Hal␣ and Hal␤ were investigated, indicating that they have similarities as well as some distinct differences compared with other two-component lantibiotics.lanthionine ͉ dehydroalanine ͉ antibiotic

Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides that are characterized by the thioether cross-linked amino acids lanthionine (Lan) and methyllanthionine (MeLan). Cinnamycin is a 19 amino acid lantibiotic that contains one Lan and two MeLan. Cinnamycin also contains an unusual lysinoalanine (Lal) bridge formed from the ε-amino group of lysine 19 and a serine residue at position 6, and an erythro-3-hydroxy-l-aspartic acid resulting from the hydroxylation of l-aspartate at position 15. These modifications are critical in mediating the interactions of cinnamycin with its target, phosphatidylethanolamine. Recently, the cinnamycin biosynthetic gene cluster (cin) from Streptomyces cinnamoneus cinnamoneus DSM 40005 was reported. Herein, we investigated the biosynthetic machinery using both in vitro studies and heterologous expression in Escherichia coli. CinX is an α-ketoglutarate/iron(II)-dependent hydroxylase that carries out the hydroxylation of aspartate 15 of the precursor peptide CinA. In addition, CinM catalyzes dehydration of four Ser and Thr residues and subsequent cyclization of Cys residues to form the three (Me)Lan bridges. The order of the post-translational modifications catalyzed by CinM and CinX is interchangeable in vitro. CinX did not require the leader sequence at the N-terminus of CinA for activity, but the leader peptide was necessary for CinM function. Although CinM dehydrated serine 6, it did not catalyze the formation of Lal. A small protein encoded by cinorf7 is critical for the formation of the cross-link between Lys19 and dehydroalanine 6 as shown by coexpression studies of CinA, CinM, CinX, and Cinorf7 in E. coli.

Lantibiotics are post-translationally modified peptide antimicrobial agents that are synthesized with an N-terminal leader sequence and a C-terminal propeptide. Their maturation involves enzymatic dehydration of Ser and Thr residues in the precursor peptide to generate unsaturated amino acids, which react intramolecularly with nearby cysteines to form cyclic thioethers termed lanthionines and methyllanthionines. The role of the leader peptide in lantibiotic biosynthesis has been subject to much speculation. In this study, mutations of conserved residues in the leader sequence of the precursor peptide for lacticin 481 (LctA) did not inhibit dehydration and cyclization by lacticin 481 synthetase (LctM) showing that not one specific residue is essential for these transformations. These amino acids may therefore be conserved in the leader sequence of class II lantibiotics to direct other biosynthetic events, such as proteolysis of the leader peptide or transport of the active compound outside the cell. However, introduction of Pro residues into the leader peptide strongly affected the efficiency of dehydration, consistent with recognition of the secondary structure of the leader peptide by the synthetase. Furthermore, the presence of a hydrophobic residue at the position of Leu-7 appears important for activity. Based on the data in this work and previous studies, a model for the interaction of LctM with LctA is proposed. The current study also showcases the ability to prepare other lantibiotics in the class II lacticin 481 family, including nukacin ISK-1, mutacin II, and ruminococcin A using the lacticin 481 synthetase. Surprisingly, a conserved Glu located in a ring that appears conserved in many class II lantibiotics, including those not belonging to the lacticin 481 subgroup, is not essential for antimicrobial activity of lacticin 481. KeywordsLantibiotic; leader peptide; lacticin 481; mutacin II; nukacin ISK-1The problem of multi-drug resistant bacteria has become increasingly apparent in recent years, with several strains posing the threat of becoming immune against all commercially available antibiotics (1,2). It is evident that in order to prevent potential epidemic outbreaks of infectious diseases, a renewed focus on antibiotic research is highly desired, including the understanding of biosynthetic pathways of natural product antibiotics (3). In this regard, the lantibiotics family of antimicrobial peptides has shown promising properties (4). Nisin, the most studied lantibiotic to date, is produced by Lactococcus lactis and has been used commercially as a preservative in the food industry for over 40 years due to its potent antibacterial properties and non-toxicity to humans (5). The high efficacy of nisin (nM MICs against many Gram-positive † This work was supported by the National Institutes of Health (GM58822 to WAV) and a Ruth L. Kirschstein National Research Service Award (GM070421 to LEC). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript bacteria) has been attributed ...

Summary The lantibiotic haloduracin consists of two post-translationally processed peptides, Halα and Halβ, that act in synergy to provide bactericidal activity. An in vitro haloduracin production system was utilized to examine the biological impact of disrupting individual thioether rings in each peptide. Surprisingly, the Halα B-ring, which contains a highly conserved CTLTXEC motif, was expendable. This motif has been proposed to interact with haloduracin’s predicted target, lipid II. Exchange of the glutamate residue in this motif for alanine or glutamine did completely abolish antibacterial activity. This study also established that Halα-Ser26 and Halβ-Ser22 escape dehydration, requiring revision of the Halβ structure previously proposed. Extracellular proteases secreted by the producer strain can remove the leader peptide, and the Halα cystine that is dispensable for bioactivity protects Halα from further proteolytic degradation.

Lantibiotics are peptide antimicrobials containing the thioether-bridged amino acids lanthionine (Lan) and methyllanthionine (MeLan) and often the dehydrated residues dehydroalanine (Dha) and dehydrobutyrine (Dhb). While biologically advantageous, the incorporation of these residues into peptides is synthetically daunting, and their production in vivo is limited to peptides containing proteinogenic amino acids. The lacticin 481 synthetase LctM offers versatile control over the installation of dehydro amino acids and thioether rings into peptides. In vitro processing of semisynthetic substrates unrelated to the prelacticin 481 peptide demonstrated the broad substrate tolerance of LctM. Furthermore, a chemoenzymatic strategy was employed to generate novel thioether linkages by cyclization of peptidic substrates containing the nonproteinogenic cysteine analogs homocysteine and beta-homocysteine. These findings are promising with respect to the utility of LctM toward preparation of conformationally constrained peptide therapeutics.