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.

With billions of years of evolution under its belt, Nature has been expanding and optimizing its biosynthetic capabilities. Chemically complex secondary metabolites continue to challenge and inspire today’s most talented synthetic chemists. A brief glance at these natural products, especially the substantial structural variation within a class of compounds, clearly demonstrates that Nature has long played the role of medicinal chemist. The recent explosion in genome sequencing has expanded our appreciation of natural product space and the vastness of uncharted territory that remains. One small corner of natural product chemical space is occupied by the recently dubbed thiazole/oxazole-modified microcins (TOMMs), which are ribosomally produced peptides with posttranslationally installed heterocycles derived from cysteine, serine and threonine residues. As with other classes of natural products, the genetic capacity to synthesize TOMMs has been widely disseminated among bacteria. Over the evolutionary timescale, Nature has tested countless random mutations and selected for gain of function in TOMM biosynthetic gene clusters, yielding several privileged molecular scaffolds. Today, this burgeoning class of natural products encompasses a structurally and functionally diverse set of molecules (i.e. microcin B17, cyanobactins, and thiopeptides). TOMMs presumably provide their producers with an ecological advantage. This advantage can include chemical weapons wielded in the battle for nutrients, disease-promoting virulence factors, or compounds presumably beneficial for symbiosis. Despite this plethora of functions, many TOMMs await experimental interrogation. This review will focus on the biosynthesis and natural combinatorial diversity of the TOMM family.

The thiazole/oxazole-modified microcins (TOMMs) represent a burgeoning class of ribosomal natural products decorated with thiazoles and (methyl)oxazoles originating from cysteines, serines and threonines. The ribosomal nature of TOMMs allows for the generation of derivative products from mutations in the amino acid sequence of the precursor peptide, which ultimately manifest in differing structures and sometimes, biological functions. Employing a TOMM system for the purpose of creating new structures and functions via combinatorial biosynthesis requires processing machinery that can tolerate highly variable substrates. In this study, TOMM enzymatic promiscuity was assessed using a currently uncharacterized cluster in Bacillus sp. Al Hakam. As determined by Fourier transform tandem mass spectrometry (FT-MS/MS), azole rings were formed in both a regio- and chemoselective fashion. Cognate and non-cognate precursor peptides were modified in an overall C- to N-terminal directionality, which to date is unique among characterized ribosomal natural products. Studies focused on the inherent promiscuity of the biosynthetic machinery elucidated a modest bias for glycine at the preceding (−1) position and a remarkable flexibility in the following (+1) position, even allowing for the incorporation of charged amino acids and bisheterocyclization. Two unnatural substrates were utilized as the conclusive test of substrate flexibility, of which both were processed in a predictable fashion. A greater understanding of substrate processing and enzymatic tolerance towards unnatural substrates will prove beneficial when designing combinatorial libraries to screen for artificial TOMMs that exhibit desired activities.

The soil dwelling, plant-growth promoting bacterium, Bacillus amyloliquefaciens FZB42, is a prolific producer of complex natural products. Recently, a new FZB42 metabolite, plantazolicin (PZN), has been described as a member of the growing thiazole/oxazole-modified microcin (TOMM) family. TOMMs are biosynthesized from inactive, ribosomal peptides and undergo a series of cyclodehydrations, dehydrogenations, and other modifications to become bioactive natural products. Using high-resolution mass spectrometry, chemoselective modification, genetic interruptions, and other spectroscopic tools, we have determined the molecular structure of PZN. In addition to two conjugated polyazole moieties, the amino-terminus of PZN has been modified to Nα,Nα-dimethylarginine. PZN exhibited a highly selective antibiotic activity towards Bacillus anthracis, but no other tested human pathogen. By altering oxygenation levels during fermentation, PZN analogs were produced that bear variability in their heterocycle content, which yielded insight into the order of biosynthetic events. Lastly, genome-mining has revealed the existence of four additional PZN-like biosynthetic gene clusters. Given their structural uniqueness and intriguing antimicrobial specificity, the PZN class of antibiotics may hold pharmacological value.

Summary Natural products are the most historically significant source of compounds for drug development. However, unacceptably high rates of compound rediscovery associated with large-scale screening of common microbial producers have resulted in the abandonment of many natural product drug discovery efforts, despite the increasing prevalence of clinically-problematic antibiotic resistance. Screening of underexplored taxa represents one strategy to avoid rediscovery. Herein we report the discovery, isolation, and structural elucidation of streptomonomicin (STM), an antibiotic lasso peptide from Streptomonospora alba, and report the genome for its producing organism. STM-resistant clones of Bacillus anthracis harbor mutations to walR, the gene encoding a response regulator for the only known widely-distributed and essential two-component signal transduction system in Firmicutes. Streptomonospora had been hitherto biosynthetically and genetically uncharacterized, with STM being the first reported compound from the genus. Our results demonstrate that understudied microbes remain fruitful reservoirs for the rapid discovery of novel, bioactive natural products.