YcaO domains use ATP to activate amide backbones during peptide cyclodehydrations

Abstract

Thiazole/oxazole-modified microcins (TOMMs) encompass a recently defined class of ribosomally synthesized natural products with a diverse set of biological activities. Although TOMM biosynthesis has been investigated for over a decade, the mechanism of heterocycle formation by the synthetase enzymes remains poorly understood. Using substrate analogs and isotopic labeling, we demonstrate that adenosine 5′-triphosphate (ATP) is utilized to directly phosphorylate the peptide amide backbone during TOMM heterocycle formation. Moreover, we present the first experimental evidence that the D-protein component of the heterocycle-forming synthetase (YcaO/DUF181 family member), formerly annotated as a docking/scaffolding protein involved in complex formation and regulation, is able to perform the ATP-dependent cyclodehydration reaction in the absence of the other TOMM biosynthetic proteins. Together, these data provide a greater level of detail into the biosynthesis of azol(in)e heterocycles in ribosomal natural products and prompt a reclassification of the enzymes involved in their installation.

Structural investigation of ribosomally synthesized natural products by hypothetical structure enumeration and evaluation using tandem MS

“…The thiazolines are subsequently dehydrogenated by the flavin mononucleotide (FMN)-containing enzyme BcerB to form thiazole heterocycles (Fig. 5A) (28,29). HSEE analysis indicated that for the intermediate containing one thiazole ring (loss of 20 Da) the structure containing a C-terminal thiazole has the highest Hs values (Fig.…”

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.…”

The Cyanobactin Heterocyclase Enzyme: A Processive Adenylase That Operates with a Defined Order of Reaction

“…[6] BalhD is homologous to the C-terminal 400 residues of TruD, whilst a second protein BalhC matches the N-terminal 300 residues ( Figure S2). When both BalhD and BalhC are present heterocyclization of the microcin substrate is accelerated and only when both proteins are present can phosphate release be robustly measured.…”

“…The immediate sequence context and position (relative to the leader) of the target cysteine is variable. a kinase type mechanism in which a hemiorthoamide intermediate is phosphorylated [6,7] ( Figure S3). The cyanobactin class of heterocyclases, exemplified by TruD, possess an almost unique combination of processivity, specificity, chemical versatility, and promiscuity, which thus makes them a fascinating subject for study.…”

“…Although dTTP does not bind in ITC experiments, we find it and GTP can catalyze heterocyclization of cysteine (Figure S5 d-f); BalhD has also been shown to use GTP. [6] The coupled NADH assay reported in the study of the adenylase enzyme AcsD [10] uses lactate dehydrogenase to oxidize NADH during the reduction of pyruvate. Pyruvate is generated by pyruvate kinase, which requires ADP.…”