Our group has focused on the discovery, biosynthesis, and mode of action determination for novel natural products (NPs). It is beyond contestation that NPs, and simple derivatives thereof, have been the most historically significant source of drug leads for the pharmaceutical industry. Beyond their use in medicine, NPs have inspired generations of synthetic chemists and provided the chemical tools to unravel fundamental aspects of cell biology. Understanding what moieties of a NP give rise to a specific property enables the rational enhancement of target-binding potency, pharmacokinetic parameters, spectrum of activity, among others. Traditional synthetic approaches for establishing structure-activity relationships (SAR) are often intractable for synthetically challenging NP scaffolds. In contrast, a properly engineered biosynthetic route could represent an operationally preferable method to establish SAR and accelerate the introduction of drug leads. This project is comprised of three related, yet fully independent, specific aims. Each proposed aim targets a unique, architecturally-complex NP scaffold that originates from a ribosomal precursor peptide. For each tar- get, we aim to reconstitute the biosynthetic pathway in vitro and generate analogs for SAR purposes by mutation of the precursor peptide gene. The mechanistic characterization of key biosynthetic enzymes will also be evaluated.
Aim I focuses on the thiazole-rich thiopeptide antibiotics, whose members inhibit different aspects of bacterial translation. The thiazoles are formed by the action of a cyclodehydratase, of which we have considerable expertise. Thiazole-forming cyclodehydratases belong to the cryptically named YcaO superfamily, which we have shown to utilize ATP in the phosphorylation of amide carbonyl oxygens. This direct amide backbone activation mechanism facilitates the cyclodehydration reaction. Thiopeptides are further defined by a central pyridine or dehydropiperidine macrocycle, arising from a predicted and chemically fascinating [4+2] cycloaddition of two dehydrated Ser residues, which this project will also fully characterize.
Aim II targets the in vitro biosynthesis of thioviridamide, a thioamide-containing, apoptosis-activatin NP.
Aim III is dedicated to the macroamidine- and thiazole-containing bottromycins, which represent an undeveloped class of bacterial translation inhibitor. All three of these biosynthetic gene clusters encode YcaO enzymes (two for bottromycin), which we implicate in roles beyond cyclodehydration. In the case of thioviridamide, the YcaO is predicted to be involved in thioamide formation while the YcaOs in bottromycin have suspected involvement in thiazole and macroamidine formation, all via an ATP-dependent amide carbonyl activation mechanism. This project will significantly advance our general understanding of NP biosynthesis and mechanistic enzymology, while also establishing the enzymatic tolerance of three NP pathways. Imparting additional chemical diversity into known NP scaffolds by our proposed heterologous and chemoenzymatic approaches holds enormous promise for revealing new drug leads and eventually expanding our pharmaceutical armamentarium.

Public Health Relevance

Natural products continue to be our most valuable source of medicine, especially when considering antibiotics. This project aims to characterize a class of enzymes involved in the synthesis of biomedically-relevant natural products. Armed with this knowledge, we will be strongly positioned to rapidly generate derivatives for drug development purposes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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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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