Insights into the Mechanism of Peptide Cyclodehydrations Achieved through the Chemoenzymatic Generation of Amide Derivatives

Abstract

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 18O-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.

Identification of an Auxiliary Leader Peptide-Binding Protein Required for Azoline Formation in Ribosomal Natural Products

“…11,24–27 The decrease in processing efficiency can often be rescued in part by the addition of the leader peptide in trans . These studies have demonstrated that, in addition to providing substrate recognition, leader peptides activate their cognate biosynthetic machineries, presumably by biasing the enzymes to an active conformation.…”

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function

“…548 The hydrolytic susceptibility of thiazolines has been exploited to introduce [ 18 O] labels into peptides for mechanistic studies (AMPL method). 183 Ring opening of thiazolines in peptides with acidic [ 18 O]-H 2 O results in the incorporation of [ 18 O] at the preceding carbonyl oxygen position (Figure 34). In contrast, thiazoles cannot be hydrolyzed by simple acid or base treatment.…”

“…YcaO domains utilize ATP directly for catalysis (section 6.2.1) 17, 183 and it has been observed that these enzymes evolved to limit the non-productive hydrolysis of ATP. For example, ATP consumption is stoichiometric (1:1) with heterocycle formation so ATP is efficiently coupled to catalysis.…”

Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

“…YcaO domain proteins activate backbone amide bonds by phosphorylation26, 27 or adenylation28 of the carbonyl oxygen, and all YcaO domain proteins with a characterized activity have a partner cyclodehydratase that aids catalysis of cyclization to oxazolines or thiazolines 29. The bottromycin gene cluster encodes two YcaO domain proteins, BtmE and BtmF, but no cyclodehydratases.…”