Kinetic Analysis of Lanthipeptide Cyclization by Substrate-Tolerant ProcM.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by the presence of thioether crosslinks. Class II lanthipeptide synthetases are bifunctional enzymes responsible for the multistep chemical modification of these natural products. ProcM is a class II lanthipeptide synthetase known for its remarkable substrate tolerance and ability to install diverse (methyl)lanthionine rings with high accuracy. Previous studies suggested that the final ring pattern of the lanthipeptide product may be determined by the substrate sequence rather than by ProcM, and that ProcM operates by a kinetically controlled mechanism, wherein the ring pattern is dictated by the relative rates of the individual cyclization reactions. This study utilizes kinetic assays to determine if rates of isolated modifications can predict the final ring pattern present in prochlorosins. Changes in the core substrate sequence resulted in changes to the reaction rates of ring formation as well as a change in the order of modifications. Additionally, individual chemical reaction rates were significantly impacted by the presence of other modifications on the peptide. These findings indicate that the rates of isolated modifications are capable of predicting the final ring pattern but are not necessarily a good predictor of the order of modification in WT ProcA3.3 and its variants.

Sequence controlled secondary structure is important for the site-selectivity of lanthipeptide cyclization.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides that are generated from precursor peptides through a dehydration and cyclization process. ProcM, a class II lanthipeptide synthetase, demonstrates high substrate tolerance. It is enigmatic that a single enzyme can catalyze the cyclization process of many substrates with high fidelity. Previous studies suggested that the site-selectivity of lanthionine formation is determined by substrate sequence rather than by the enzyme. However, exactly how substrate sequence contributes to site-selective lanthipeptide biosynthesis is not clear. In this study, we performed molecular dynamic simulations for ProcA3.3 variants to explore how the predicted solution structure of the substrate without enzyme correlates to the final product formation. Our simulation results support a model in which the secondary structure of the core peptide is important for the final product's ring pattern for the substrates investigated. We also demonstrate that the dehydration step in the biosynthesis pathway does not influence the site-selectivity of ring formation. In addition, we performed simulation for ProcA1.1 and 2.8, which are well-suited candidates to investigate the connection between order of ring formation and solution structure. Simulation results indicate that in both cases, C-terminal ring formation is more likely which was supported by experimental results. Our findings indicate that the substrate sequence and its solution structure can be used to predict the site-selectivity and order of ring formation, and that secondary structure is a crucial factor influencing the site-selectivity. Taken together, these findings will facilitate our understanding of the lanthipeptide biosynthetic mechanism and accelerate bioengineering efforts for lanthipeptide-derived products.

Improved production of class I lanthipeptides in Escherichia coli.

Lanthipeptides are ribosomally synthesised and post-translationally modified peptides containing lanthionine (Lan) and methyllanthionine (MeLan) residues that are formed by dehydration of Ser/Thr residues followed by conjugate addition of Cys to the resulting dehydroamino acids. Class I lanthipeptide dehydratases utilize glutamyl-tRNAGlu as a co-substrate to glutamylate Ser/Thr followed by glutamate elimination. Here we report a new system to heterologously express class I lanthipeptides in Escherichia coli through co-expression of the producing organism's glutamyl-tRNA synthetase (GluRS) and tRNAGlu pair in the vector pEVOL. In contrast to the results in the absence of the pEVOL system, we observed the production of fully-dehydrated peptides, including epilancin 15X, and peptides from the Bacteroidota Chryseobacterium and Runella. A second common obstacle to production of lanthipeptides in E. coli is the formation of glutathione adducts. LanC-like (LanCL) enzymes were previously reported to add glutathione to dehydroamino-acid-containing proteins in Eukarya. Herein, we demonstrate that the LanCL enzymes can remove GSH adducts from C-glutathionylated peptides with dl- or ll-lanthionine stereochemistry. These two advances will aid synthetic biology-driven genome mining efforts to discover new lanthipeptides.

Mechanism of Action of Ribosomally Synthesized and Post-Translationally Modified Peptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a natural product class that has undergone significant expansion due to the rapid growth in genome sequencing data and recognition that they are made by biosynthetic pathways that share many characteristic features. Their mode of actions cover a wide range of biological processes and include binding to membranes, receptors, enzymes, lipids, RNA, and metals as well as use as cofactors and signaling molecules. This review covers the currently known modes of action (MOA) of RiPPs. In turn, the mechanisms by which these molecules interact with their natural targets provide a rich set of molecular paradigms that can be used for the design or evolution of new or improved activities given the relative ease of engineering RiPPs. In this review, coverage is limited to RiPPs originating from bacteria.