Site-Specific Nonenzymatic Peptide S/O-Glutamylation Reveals the Extent of Substrate Promiscuity in Glutamate Elimination Domains

Formation of dehydroalanine
and dehydrobutyrine residues via tRNA-dependent
dehydration of serine and threonine is a key post-translational modification
in the biosynthesis of lanthipeptide and thiopeptide RiPPs. The dehydration
process involves two reactions, wherein the O-glutamyl Ser/Thr intermediate,
accessed by a dedicated enzyme utilizing Glu-tRNAGlu as
the acyl donor, is recognized by the second enzyme, referred to as
the glutamate elimination domain (ED), which catalyzes the eponymous
reaction yielding a dehydroamino acid. Many details of ED catalysis
remain unexplored because the scope of available substrates for testing
is limited to those that the upstream enzymes can furnish. Here, we
report two complementary strategies for direct, nonenzymatic access
to diverse ED substrates. We establish that a thiol-thioester exchange
reaction between a Cys-containing peptide and an α thioester of glutamic acid leads an S-glutamylated intermediate
which can act as a substrate for EDs. Furthermore, we show that the
native O-glutamylated substrates can be accessible from S-glutamylated
peptides upon a site-specific S-to-O acyl transfer reaction. Combined
with flexible in vitro translation utilized for rapid peptide production,
these chemistries enabled us to dissect the substrate recognition
requirements of three known EDs. Our results establish that EDs are
uniquely promiscuous enzymes capable of acting on substrates with
arbitrary amino acid sequences and performing retro-Michael reaction
beyond the canonical glutamate elimination. To facilitate substrate
recruitment, EDs apparently engage in nonspecific hydrophobic interactions
with their substrates. Altogether, our results establish the substrate
scope of EDs and provide clues to their catalysis.