Pseudokinases can catalyse peptide cyclization through thioether crosslinking.

The protein-kinase-like superfamily proteins are crucial and generally catalyse substrate phosphorylation using adenosine 5'-triphosphate. Pseudokinases are non-canonical protein-kinase-like members deficient in kinase activity, and few of them are known to be enzymatically active and to have catalytic ability rather than phosphorylation. Based on biosynthetic investigations into thioamitides and lanthipeptides-two different families of ribosomally synthesized and post-translationally modified peptides-we here report a peptide cyclization activity of pseudokinases (TvaE and SacE) that enables (ene)thioether residue formation. We determine the dedicated cyclase activity in unsaturated 2-aminovinyl-cysteine formation and mine for similar activity in saturated lanthionine formation. Biochemical characterization, heterologous expression, co-crystallization, computational analysis, genome mining, isotope labelling and site-specific mutagenesis rationalize the commonality in catalysis, demonstrating that a protein-kinase fold can be repurposed for unexpected utilities. Related cyclases differ from known enzymatically active pseudokinases that resemble canonical protein-kinase-like proteins in mechanism and function. Instead, they catalyse Michael addition for (ene)thioether crosslinking through a sandwich-like substrate-assisted process.