An Orthogonal D2O‐Based Induction System that Provides Insights into d‐Amino Acid Pattern Formation by Radical S‐Adenosylmethionine Peptide Epimerases

Abstract

AbstractRadical S‐adenosyl methionine peptide epimerases (RSPEs) are an enzyme family that accomplishes regiospecific and irreversible introduction of multiple d‐configured residues into ribosomally encoded peptides. Collectively, RSPEs can generate diverse epimerization patterns in a wide range of substrates. Previously, the lack of rapid methods to localize epimerized residues has impeded efforts to investigate the function and applicative potential of RSPEs. An efficient mass spectrometry‐based assay is introduced that permits characterization of products generated in E. coli. Applying this to a range of non‐natural peptide‐epimerase combinations, it is shown that the d‐amino acid pattern is largely but not exclusively dictated by the core peptide sequence, while the epimerization order is dependent on the enzyme‐leader pair. RSPEs were found to be highly promiscuous, which allowed for modular introduction of peptide segments with defined patterns.