Macroamidine Formation in Bottromycins Is Catalyzed by a Divergent YcaO Enzyme

Abstract

The YcaO superfamily of proteins catalyzes the phosphorylation of peptide backbone amide bonds, which leads to the formation of azolines and azoles in ribosomally synthesized and post-translationally modified peptides (RiPPs). Bottromycins are RiPPs with potent antimicrobial activity, and their biosynthetic pathway contains two divergent, stand-alone YcaO enzymes, IpoC and PurCD. From an untargeted metabolomics approach, it had been suggested that PurCD acts with a partner protein to form the 12-membered macroamidine unique to bottromycins. Here we report the biochemical characterization of IpoC and PurCD. We demonstrate that IpoC installs a cysteine-derived thiazoline, whereas PurCD alone is sufficient to create the macroamidine structure. Both enzymes are catalytically promiscuous, and we generated 10 different macroamidines. Our data provide important insights into the versatility of YcaO enzymes, their ability to utilize different nucleophiles and provide a framework for the creation of novel bottromycin derivatives with enhanced bioactivity.

Discovery and characterisation of an amidine-containing ribosomally-synthesised peptide that is widely distributed in nature

“…The BGC for bottromycin encodes two YcaO proteins, where one is required for macroamidine formation [29] and the other catalyses heterocyclisation of a cysteine residue to a thiazoline; both function without a partner protein. [14,30] In the case of streptamidine, the gene deletion data are consistent with a role in cyclisation for AmiB and the YcaO protein, AmiD. Conventional sequence analysis did not identify any conserved domains for AmiB, but structural modelling with Phyre2 [31] predicts that it has a homologous structure to residues 4-315 of the cyanobactin heterocyclase TruD, [32] encompassing a RiPP recognition element [33] and an E1-like domain.…”

“…This includes the installation of oxazoline and thiazoline heterocycles onto the precursor peptide backbone, where cyclodehydration is catalysed by the YcaO-domain in cooperation with a protein homologous to an E1 ubiquitin-activation enzyme or an "Ocin-ThiF-like" protein. [13] YcaO proteins have also been demonstrated to catalyse the formation of amidine rings in bottromycin [14] and klebsazolicin, [15] and can also function with a TfuA-domain protein to introduce thioamide bonds into RiPPs such as thiopeptin [16] and the thiovarsolins. [7,17] Over 9,000 YcaO-domain proteins have been bioinformatically identified in GenBank, but the function of the majority of these remain unknown.…”

Regulation of Bottromycin Biosynthesis Involves an Internal Transcriptional Start Site and a Cluster-Situated Modulator

“…Bottromycin is a ribosomally synthesized and post-translationally modified peptide (RiPP). In its biosynthetic pathway, a precursor peptide (BtmD) undergoes a complex and unprecedented series of modifications catalyzed by enzymes encoded in the bottromycin (btm) gene cluster (Figure 1) (Crone et al, 2012(Crone et al, , 2016Gomez-Escribano et al, 2012;Huo et al, 2012;Franz et al, 2017;Schwalen et al, 2017;Sikandar et al, 2019).…”

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool

“…15 These were originally shown to be responsible for the introduction of oxazoline and thiazoline heterocycles in the PP backbone of microcins, 16 and were very recently demonstrated to catalyze the formation of the macroamidine ring of bottromycin. [17][18][19] YcaO proteins act as cyclodehydratases, activating the amide bond substrate by nucleophilic attack, which is followed by ATP-driven O-phosphorylation of the hemiorthoamide intermediate and subsequent elimination of phosphate. In most azoline-containing RiPPs, this catalytic activity requires a partner protein (E1-like or Ocin-ThiF-like proteins that are clustered with or fused to the YcaO domain), which acts as a docking element to bring the precursor peptide to the active site of the cyclodehydratase.…”

“…YcaO proteins can also act as standalone proteins, as in bottromycin biosynthesis, [17][18][19] and many YcaO proteins are encoded in genomes without E1-like or Ocin-ThiF-like partner proteins, 9,15 including in the BGCs of thioviridamide-like molecules. 6,[20][21][22][23][24] Thioviridamide and related compounds are cytotoxic RiPPs that contain multiple thioamide groups ( Figure 1), but no azole or macroamidine rings.…”