title: 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.
authors: Department of Chemistry and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.
authors: Subha
authors: S
authors: Mukherjee
authors: Wilfred A
authors: WA
authors: van der Donk
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000737:chemistry; D055162:Biocatalysis; D003500:Cyclization; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000737:chemistry; D048929:Prochlorococcus / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D060327:Solid-Phase Synthesis Techniques; D013379:Substrate Specificity; D013440:Sulfides / Q000737:chemistry*
chemical_list: D001430:Bacteriocins; D010456:Peptides, Cyclic; D013440:Sulfides; D008025:Ligases; C001520:lanthionine; D000409:Alanine