| Lantibiotics are post-translationally modified antimicrobial peptides containing multiple cyclic thioether-containing amino acids, termed lanthionines (Lan), that constrain their conformational freedom and that are required for their potent antimicrobial properties. These compounds also typically contain unsaturated amino acids, such as dehydroalanine (Dha) and dehydrobutyrine (Dhb). These nonproteinogenic amino acids are installed in a two-step enzymatic process that involves dehydration of Ser or Thr residues in the substrate prepeptide (LanA) to the corresponding Dha and Dhb. This is followed by a subsequent conjugate addition by Cys thiols onto the dehydro amino acids to provide Lan or methyllanthionine (MeLan) rings. In Class I lantibiotics, dehydration and cyclization are catalyzed by dedicated LanB and LanC enzymes, respectively, whereas a bifunctional LanM enzyme catalyzes both of these reactions for Class II lantibiotics. The activity of the bifunctional enzyme, lacticin 481 synthetase (LctM), required for the biosynthesis of the Class II 'antibiotic lacticin 481, was recently reconstituted in vitro.;The substrate specificity of LctM in vitro was investigated using truncated LctA substrates containing a variety of nonproteinogenic amino acids. Insights gained from these studies were utilized to guide the synthesis of a variety of lacticin 481 analogues containing nonproteinogenic amino acids, including D-amino acids, beta-amino acids, and N -alkyl glycine (peptoid) residues. The bioactivity of these unnatural lacticin 481 analogues was evaluated against two bacterial strains using agar diffusion assays. A few analogues were identified that displayed higher activity than the parent compound. In the midst of these studies, new methodology was developed for the synthesis of triazole-linked LctA prepeptides.;The promiscuous activity of LctM toward LctA prepeptides containing nonproteinogenic amino acids prompted the evaluation of LctM as a general catalyst for the introduction of post-translational modifications in non-lantibiotic peptides fused to the LctA leader peptide. LctM was utilized to efficiently introduce dehydrated, phosphorylated, and lanthionine cross-linked amino acids into therapeutically relevant non-lantibiotic peptides. Furthermore, enzymatically installed dehydro amino acids were used as sites of ligation with a variety of thiol nucleophiles for the preparation of peptide conjugates. Lastly, the role of the leader peptide in lacticin 481 biosynthesis was investigated. Surprisingly, the leader peptide was not required for dehydration activity of LctM, although it greatly enhanced the catalytic efficiency of LctM. |
