Mechanistic Studies on the Substrate-Tolerant Lanthipeptide Synthetase ProcM

Abstract

Lanthipeptides are a class of post-translationally modified peptide natural products. They contain lanthionine (Lan) and methyllanthionine (MeLan) residues, which generate cross-links and endow the peptides with various biological activities. The mechanism of a highly substrate-tolerant lanthipeptide synthetase, ProcM, was investigated herein. We report a hybrid ligation strategy to prepare a series of substrate analogues designed to address a number of mechanistic questions regarding catalysis by ProcM. The method utilizes expressed protein ligation to generate a C-terminal thioester of the leader peptide of ProcA, the substrate of ProcM. This thioester was ligated with a cysteine derivative that resulted in an alkyne at the C-terminus of the leader peptide. This alkyne in turn was used to conjugate the leader peptides to a variety of synthetic peptides by copper-catalyzed azide–alkyne cycloaddition. Using deuterium-labeled Ser and Thr in the substrate analogues thus prepared, dehydration by ProcM was established to occur from C-to-N-terminus for two different substrates. Cyclization also occurred with a specific order, which depended on the sequence of the substrate peptides. Furthermore, using orthogonal cysteine side-chain protection in the two semisynthetic peptide substrates, we were able to rule out spontaneous non-enzymatic cyclization events to explain the very high substrate tolerance of ProcM. Finally, the enzyme was capable of exchanging protons at the α-carbon of MeLan, suggesting that ring formation could be reversible. These findings are discussed in the context of the mechanism of the substrate-tolerant ProcM, which may aid future efforts in lanthipeptide engineering.

Structure and tRNA Specificity of MibB, a Lantibiotic Dehydratase from Actinobacteria Involved in NAI-107 Biosynthesis

“…Our data then implies Ser5 to be dehydrated last. Based on the collected information MibB dehydrates the precursor peptide MibA without strict directionality (Figure S2C), an observation that has also been reported for other lanthipeptide biosynthetic systems (Jungmann et al, 2014, Mukherjee and van der Donk, 2014). …”

Product Formation by the Promiscuous Lanthipeptide Synthetase ProcM is under Kinetic Control

“…A ProcA3.3 derivative with a Cys21-Dhb18 ring was generated by orthogonal protection of Cys14 using methodology previously described (Figure 8). 9 Incubation of the intermediate with WT ProcM did not result in conversion of this “wrong intermediate” to the correct product with overlapping rings, but instead a product with non-overlapping rings was formed (Figures 8 and S14). In other words, once the incorrect first ring was installed, ProcM proceeded to the incorrect product.…”

“…The order of dehydration has been established in a previous study for ProcA2.8 and ProcA3.3. 9 To study cyclization in isolation, the putative cyclase domains of WT ProcM and ProcM-C971H were obtained by expressing the C-terminal domains spanning residues 655-1068 (ProcM-655-1068 and ProcM-C971H-655-1068). The cyclization reaction catalyzed by ProcM-655-1068 was studied with dehydrated ProcA3.3 that was generated as described in the previous section.…”

Pseudomycoicidin, a Class II Lantibiotic from Bacillus pseudomycoides

“…In the experiments with the mutant peptides, the modification by PseM was disturbed by the removal of Cys residues, indicating that the substrate specificity of PseM is much narrower than that of NisB (60), LctM (61), or especially ProcM (62). This also was confirmed when we tried to coexpress another predicted lantibiotic structural gene of Caldicellulosiruptor bescii (40) fused to the pseudomycoicidin leader along with PseM in E. coli, which did not result in modification of the core peptide.…”