Mechanistic Insights Into Post‐translational α‐Keto‐β‐Amino Acid Formation by a Radical S‐Adenosyl Methionine Peptide Splicease

Abstract

Radical S‐adenosyl methionine enzymes catalyze a diverse repertoire of post‐translational modifications in protein and peptide substrates. Among these, an exceptional and mechanistically obscure example is the installation of α‐keto‐β‐amino acid residues by formal excision of a tyrosine‐derived tyramine unit. The responsible spliceases are key maturases in a widespread family of natural products termed spliceotides that comprise potent protease inhibitors, with the installed β‐residues being crucial for bioactivity. Here, we established the in vitro activity of the model splicease PcpXY to interrogate the mechanism of non‐canonical protein splicing. Identification of shunt and coproducts, deuterium labeling studies, and density functional theory energy calculations of hypothesized intermediates support a mechanism involving hydrogen abstraction at tyrosine Cα as the initial site of peptide radical formation and release of 4‐hydroxybenzaldehyde as the tyrosine‐derived coproduct. The data illuminate key features of this unprecedented radical‐mediated biotransformation yielding ketoamide pharmacophores that are also present in peptidomimetic therapeutics.