# Characterization of YcaO-Dependent Natural Product Biosynthetic Pathways > **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN · 2020 · $337,662 ## Abstract Our group is broadly interested in the chemical biology of natural products with a strong focus on genomics- based discovery, biosynthetic mechanistic enzymology, and determination of structure-activity relationships and mode of action. Beyond their historical impact on medicine, natural products have inspired generations of syn- thetic chemists and provided the necessary chemical probes to illuminate fundamental aspects of biology. One natural product family that has received increased attention over the past several years are the ribosomally synthesized and post-translationally modified peptides (RiPPs). While there are over 30 distinct structural clas- ses of RiPP natural products reported, they are united by a common biosynthetic logic: a precursor peptide, typically composed of an N-terminal leader and a C-terminal core, is ribosomally produced. The leader region contains motifs that are recognized by the modification enzymes and the core region is where the modifications take place. Upon maturation, the leader region is often removed prior to cellular export. The current project focuses on natural product biosynthetic pathways that encode a member of the YcaO superfamily. During the original funding period, we showed that YcaO enzymes were responsible for the ATP- dependent activation of the peptide backbone to yield azoline heterocycles from Cys, Ser, and Thr residues of the core peptide. During the current funding period, we discovered that two additional reaction types are cata- lyzed by YcaO enzymes: thioamidation and macrolactamidation of the peptide backbone. No fewer than five classes of RiPPs are now known to utilize a member of the YcaO superfamily, namely the linear azol(in)e- containing peptides, thiopeptides, cyanobactins, bottromycins, and thioviridamides. Despite a wealth of knowledge, we can only predict the modification type of approximately one-third of the YcaO superfamily. Our bioinformatics analysis suggests that several new reaction types remain to be discovered. For this renewal project, we tackle several outstanding questions with respect to YcaO-dependent natural product biosynthesis. Aim I focuses on the structurally and enzymatically intriguing thiopeptide RiPP class. Aim IA will overcome biosynthetic bottlenecks with respect to substrate tolerance in order to establish the elusive structure-activity relationships and generate advanced biosynthetic intermediates that will enable the study of late-stage transformations found within the class. Aim IB will determine the enzymatic mechanism and substrate scope of the class-defining [4+2]-cycloaddition and establish why some are pyridine-forming while others are dehydropiperidine-forming. Aim II focuses on peptide backbone thioamidation, in particular, deciphering the func- tion of the TfuA partner protein and a novel desulfurase/lysase involved in mobilizing sulfur from Cys. Lastly, Aim III characterizes divergent YcaO family members that appear in unique genomic con... ## Key facts - **NIH application ID:** 9986387 - **Project number:** 2R01GM097142-09 - **Recipient organization:** UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN - **Principal Investigator:** Douglas Alan Mitchell - **Activity code:** R01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2020 - **Award amount:** $337,662 - **Award type:** 2 - **Project period:** 2012-02-01 → 2024-07-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/9986387 ## Citation > US National Institutes of Health, RePORTER application 9986387, Characterization of YcaO-Dependent Natural Product Biosynthetic Pathways (2R01GM097142-09). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/9986387. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*