Discovery of Unique Lanthionine Synthetases Reveals New Mechanistic and Evolutionary Insights

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Abstract
Lantibiotic synthetases are remarkable biocatalysts generating conformationally constrained peptides with a variety of biological activities by repeatedly utilizing two simple posttranslational modification reactions: dehydration of Ser/Thr residues and intramolecular addition of Cys thiols to the resulting dehydro amino acids. Since previously reported lantibiotic synthetases show no apparent homology with any other known protein families, the molecular mechanisms and evolutionary origin of these enzymes are unknown. In this study, we present a novel class of lanthionine synthetases, termed LanL, that consist of three distinct catalytic domains and demonstrate in vitro enzyme activity of a family member from Streptomyces venezuelae. Analysis of individually expressed and purified domains shows that LanL enzymes install dehydroamino acids via phosphorylation of Ser/Thr residues by a protein kinase domain and subsequent elimination of the phosphate by a phosphoSer/Thr lyase domain. The latter has sequence homology with the phosphothreonine lyases found in various pathogenic bacteria that inactivate host mitogen activated protein kinases. A LanC-like cyclase domain then catalyzes the addition of Cys residues to the dehydro amino acids to form the characteristic thioether rings. We propose that LanL enzymes have evolved from stand-alone protein Ser/Thr kinases, phosphoSer/Thr lyases, and enzymes catalyzing thiol alkylation. We also demonstrate that the genes for all three pathways to lanthionine-containing peptides are widespread in Nature. Given the remarkable efficiency of formation of lanthionine-containing polycyclic peptides and the latter's high degree of specificity for their cognate cellular targets, it is perhaps not surprising that (at least) three distinct families of polypeptide sequences have evolved to access this structurally and functionally diverse class of compounds. Many bacteria generate cyclic peptides, which have improved biological activities compared to linear peptides, including higher stability. Lanthionine-containing peptides are one such group, and different members of this group have antibiotic, anti-inflammatory, and anti-viral activities. For example, one lanthionine-containing peptide called nisin has been used to protect food items from harmful bacteria. Two different pathways for the biosynthesis of lanthionine-containing peptides have been described previously. By comparing the DNA sequences of bacterial genomes we reveal a third biosynthetic route that provides further insight into how the biosynthetic pathways for these cyclic peptides have evolved. We characterized the novel lanthionine synthetase utilized in this third pathway in the soil bacterium Streptomyces venezuelae and show that the purified enzyme catalyzes the chemical reactions necessary to turn a linear peptide into a peptide with multiple rings. The discovery of this third biosynthetic pathway widens the scope for the engineering of new lanthionine-containing peptides for potential use in human therapeutics.