Highlightsd Temporal assembly of a GPCR-Gs complex revealed by time-resolved mass spectrometry d The sequence of GPCR-mediated G protein activation was elucidated d Key structural elements were found to dictate nucleotide release
Ribosomally
synthesized and post-translationally modified peptides
(RiPPs) are a growing family of bioactive peptides. Among RiPPs, the
bacterial toxin polytheonamide A is characterized by a unique set
of post-translational modifications catalyzed by novel radical S-adenosyl-l-methionine (SAM) enzymes. Here we
show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide
epimerization in a C-to-N directional
manner. By combining mutagenesis experiments with labeling studies
and investigating the enzyme substrate promiscuity, we deciphered
in detail the mechanism of PoyD. We notably identified a critical
cysteine residue as a likely key H atom donor and demonstrated that
PoyD belongs to a distinct family of radical SAM peptidyl epimerases.
In addition, our study shows that the core peptide directly influences
the epimerization pattern allowing for production of peptides with
unnatural epimerization patterns.
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