Site-specific nonenzymatic peptide S/O–glutamylation reveals the extent of substrate promiscuity in glutamate elimination domains

Abstract

Formation of dehydroalanine and dehydrobutyrine residues via tRNA-dependent dehydration of serine and threonine is a key post-translational modification in the biosynthesis of lanthipeptides and thiopeptides. The dehydration process involves two reactions, wherein the O-glutamyl Ser/Thr intermediate, accessed by a dedicated enzyme utilizing Glu-tRNA Glu 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. Owing to the fact that many lanthipeptides and thiopeptides possess strong antibiotic activities, the enzymology of their biosynthesis became the focus of numerous studies, and although significant progress has recently been made, much remains to be elucidated. This is especially true for the downstream EDs, 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. First, we establish that a thiol-thioester exchange reaction between a Cyscontaining 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 aid in the substrate recruitment process, EDs apparently engage in nonspecific hydrophobic interactions with their substrates. Altogether, our results establish the substrate scope of EDs and provide clues to their modes of action.