Pseudokinases can catalyse peptide cyclization through thioether crosslinking

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Protein-kinase-like superfamily proteins are vital for cellular signaling via phosphorylation.
• Pseudokinases are related proteins lacking canonical kinase activity, with some exhibiting unique catalytic functions.
• Ribosomally synthesized and post-translationally modified peptides, such as thioamitides and lanthipeptides, involve complex modifications.

Purpose of the Study:

• To investigate the enzymatic activity of pseudokinases beyond phosphorylation.
• To identify and characterize the peptide cyclization activity of specific pseudokinases (TvaE and SacE).
• To explore the mechanism of (ene)thioether residue formation catalyzed by these pseudokinases.

Main Methods:

• Biosynthetic investigations of thioamitides and lanthipeptides.
• Biochemical characterization and heterologous expression of pseudokinases.
• Co-crystallization, computational analysis, and site-specific mutagenesis.
• Genome mining and isotope labeling studies.

Main Results:

• Pseudokinases TvaE and SacE were found to possess peptide cyclization activity, forming (ene)thioether residues.
• The study identified dedicated cyclase activity in unsaturated 2-aminovinyl-cysteine formation and explored saturated lanthionine formation.
• A common catalytic mechanism involving Michael addition for crosslinking was elucidated, distinct from canonical protein kinases.

Conclusions:

• The protein-kinase fold can be repurposed for novel catalytic functions, such as peptide cyclization.
• Pseudokinases can act as cyclases, catalyzing Michael addition for thioether crosslinking.
• This finding expands the known functional repertoire of pseudokinases and protein-kinase-like proteins.