Kinetic Analysis of Cyclization by the Substrate-Tolerant Lanthipeptide Synthetase ProcM.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by the presence of thioether cross-links called lanthionine and methyllanthionine, formed by dehydration of Ser/Thr residues and Michael-type addition of Cys side chains onto the resulting dehydroamino acids. Class II lanthipeptide synthetases are bifunctional enzymes responsible for both steps, thus generating macrocyclic natural products. ProcM is part of a group of class II lanthipeptide synthetases that are known for their remarkable substrate tolerance, having large numbers of natural substrates with highly diverse peptide sequences. They install multiple (methyl)lanthionine rings with high accuracy, attributes that have been used to make large libraries of polycyclic peptides. Previous studies suggested that the final ring pattern of the lanthipeptide product may be determined by the substrate sequence rather than by ProcM. The current investigation on the ProcM-catalyzed modification of one of its 30 natural substrates (ProcA3.3) and its sequence variants utilizes kinetic assays to understand the factors that determine the ring pattern. The data show that changes in the substrate sequence result in changes to the reaction rates of ring formation that in some cases lead to a change in the order of the modifications and thereby bring about different ring patterns. These observations provide further support that the substrate sequence determines to a large degree the final ring pattern. The data also show that similar to a previous study on another substrate (ProcA2.8), the reaction rates of successive reactions slow down as the peptide is matured; rate constants observed for the reactions of these two substrates are similar, suggesting that they reflect the intrinsic activity of the enzyme with its 30 natural substrates. We also investigated whether rates of formation of single isolated rings can predict the final ring pattern of polycyclic products, an important question for the products of genome mining exercises, as well as library generation. Collectively, the findings in this study indicate that the rates of isolated modifications can be used for predicting the final ProcM-produced ring pattern, but they also revealed limitations. One unexpected observation was that even changing Ser to Thr and vice versa, a common means to convert lanthionine to methyllanthionine and vice versa, can result in a change in the ring pattern.