The genomics revolution has repeatedly demonstrated that our understanding of natural product (NP) biosynthesis is far from complete. Given the frequency of silent and cryptic biosynthetic clusters in existing and emerging genomes, compounded with the inability to culture >99% of microbes, far less than 1% of microbial NPs have been discovered. This project proposes to characterize novel NP biosynthetic clusters from two soil-dwelling bacteria. Without question, NPs from soil bacteria are our most prolific source of medicine. Unlocking the chemical structure and biological function of novel NPs encoded by these organisms holds enormous potential for expanding our pharmaceutical repertoire. The biosynthetic clusters of interest to this proposal are members of a recently described, evolutionarily conserved family dubbed the thiazole/oxazole-modified microcins (TOMM). As a new NP family, TOMMs represent an underexplored area of NP chemical space - few have a known structure or mechanism of action. All TOMMs with a known activity function as toxins, making them of paramount interest to modern medicine. In two known cases, TOMMs produced by human pathogens play a critical role in the molecular mechanism of pathogenesis. Therefore, a more complete knowledge of the biosynthetic pathway could lead to the development of virulence-targeting antibiotics, which represents a longer-term objective for our research program. To effectively tap into this potential, several gaps in our current understanding of these molecules must be addressed. This project is divided into three related, but independent specific aims.
For Aim 1, a combination of in vitro reconstitution, natural product isolation, and advanced spectroscopy will be employed to determine the chemical structure of the TOMM product.
In Aim 2, high-resolution mass spectrometry and site-directed mutagenesis will be used to kinetically evaluate a key enzyme that catalyzes the first step in the formation of thiazoles and oxazoles. This enzyme, a cyclodehydratase, is responsible for recognizing the TOMM precursor peptide and converting Cys and Ser/Thr residues into thiazolines and (methyl) oxazolines.
Aim 3 seeks to reveal the protein-protein interactions that enable substrate recognition and the downstream thiazole/oxazole forming activity. By characterizing the enzymes involved in TOMM biosynthesis, the foundation for future work will be laid, including the development of biosynthetic inhibitors of TOMMs from human pathogens and strategies to harness the power of combinatorial biosynthesis to evolve TOMMs with desired biological targets. Progress on this project will fill a major void in our current understanding of how a subset of peptide-derived toxins is biosynthesized. The tools developed will be broadly applicable to the study of other TOMMs.

Public Health Relevance

The majority of medicines used today are derived from complex chemicals produced by nature. Such natural products continue to be our most valuable source of new drugs. To lay the foundation for expanding the current repertoire of pharmaceuticals, we seek to characterize how bacteria synthesize a new subclass of natural products.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function E Study Section (MSFE)
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Gerratana, Barbara
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University of Illinois Urbana-Champaign
Schools of Arts and Sciences
United States
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Mahanta, Nilkamal; Liu, Andi; Dong, Shihui et al. (2018) Enzymatic reconstitution of ribosomal peptide backbone thioamidation. Proc Natl Acad Sci U S A 115:3030-3035
Schwalen, Christopher J; Hudson, Graham A; Kille, Bryce et al. (2018) Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics. J Am Chem Soc 140:9494-9501
Hudson, Graham A; Mitchell, Douglas A (2018) RiPP antibiotics: biosynthesis and engineering potential. Curr Opin Microbiol 45:61-69
Si, Tong; Li, Bin; Comi, Troy J et al. (2017) Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J Am Chem Soc 139:12466-12473
Cogan, Dillon P; Hudson, Graham A; Zhang, Zhengan et al. (2017) Structural insights into enzymatic [4+2] aza-cycloaddition in thiopeptide antibiotic biosynthesis. Proc Natl Acad Sci U S A 114:12928-12933
Nayak, Dipti D; Mahanta, Nilkamal; Mitchell, Douglas A et al. (2017) Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea. Elife 6:
Burkhart, Brandon J; Kakkar, Nidhi; Hudson, Graham A et al. (2017) Chimeric Leader Peptides for the Generation of Non-Natural Hybrid RiPP Products. ACS Cent Sci 3:629-638
Zhang, Zhengan; Mahanta, Nilkamal; Hudson, Graham A et al. (2017) Mechanism of a Class C Radical S-Adenosyl-l-methionine Thiazole Methyl Transferase. J Am Chem Soc 139:18623-18631
Mahanta, Nilkamal; Zhang, Zhengan; Hudson, Graham A et al. (2017) Reconstitution and Substrate Specificity of the Radical S-Adenosyl-methionine Thiazole C-Methyltransferase in Thiomuracin Biosynthesis. J Am Chem Soc 139:4310-4313
Schwalen, Christopher J; Hudson, Graham A; Kosol, Simone et al. (2017) In Vitro Biosynthetic Studies of Bottromycin Expand the Enzymatic Capabilities of the YcaO Superfamily. J Am Chem Soc 139:18154-18157

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