Insights into the mechanism of peptide cyclodehydrations achieved through the chemoenzymatic generation of amide derivatives.

Current strategies for generating peptides and proteins bearing amide carbonyl derivatives rely on solid-phase peptide synthesis for amide functionalization. Although such strategies have been successfully implemented, technical limitations restrict both the length and sequence of the synthetic fragments. Herein we report the repurposing of a thiazole/oxazole-modified microcin (TOMM) cyclodehydratase to site-specifically install amide backbone labels onto diverse peptide substrates, a method we refer to as azoline-mediated peptide backbone labeling (AMPL). This convenient chemoenzymatic strategy can generate both thioamides and amides with isotopically labeled oxygen atoms. Moreover, we demonstrate the first leader peptide-independent activity of a TOMM synthetase, circumventing the requirement that sequences of interest be fused to a leader peptide for modification. Through bioinformatics-guided site-directed mutagenesis, we also convert a strictly dehydrogenase-dependent TOMM azole synthetase into an azoline synthetase. This vastly expands the spectrum of substrates modifiable by AMPL by allowing any in vitro reconstituted TOMM synthetase to be employed. To demonstrate the utility of AMPL for mechanistic enzymology studies, an (18)O-labeled substrate was generated to provide direct evidence that cyclodehydrations in TOMMs occur through the phosphorylation of the carbonyl oxygen preceding the cyclized residue. Furthermore, we demonstrate that AMPL is a useful tool for establishing the location of azolines both on in vitro modified peptides and azoline-containing natural products.