# Understanding How Nonheme Iron-Dependent Enzymes Assemble Pharmacophores: Studies of Cyclopropane, Aziridine, and Isonitrile Formation

> **NIH NIH R01** · NORTH CAROLINA STATE UNIVERSITY RALEIGH · 2024 · $418,445

## Abstract

Project Summary/Abstract
Late-stage functionalization is a powerful strategy used in nature and in synthetic chemistry to expand structural
complexity and diversity. In natural product biosynthesis, this strategy is exemplified by diverse reactions
catalyzed by metalloenzymes. Among them, non-heme-iron- and 2-oxoglutarate-dependent (Fe/2OG) enzymes
have been shown to perform a staggering array of transformations including carbo- and hetero-cyclization,
halogenation, hydroxylation, olefination, and rearrangement. The catalytic diversity makes Fe/2OG enzymes an
attractive target for biocatalyst development, organic synthesis, and other applications. However, despite the
prevalence of Fe/2OG enzymes in all kingdoms of life (>160,000 encoded Fe/2OG enzyme genes in genomic
data), efficient and precise strategies to annotate Fe/2OG enzyme functions are still lacking. Cyclopropane and
aza-cyclopropane (aziridine) are unique three-membered carbo-/hetero-cycles, and are important
pharmacophores widely used in drugs. In addition, the isocyano (isonitrile) group is found in hundreds of
secondary metabolites with roles in metal trafficking. As demonstrated in the preliminary results, our combined
bioinformatics and retro-biosynthetic analysis has identified Fe/2OG cyclopropanases, aziridinases and
isonitrilases from thousands of functionally unannotated Fe/2OG enzymes. In this project, we will continue to
apply this strategy to identify enzymes with these targeted activities. In cyclopropanation and aziridination, these
newly discovered Fe/2OG enzymes and the related Fe/2OG hydroxylases will be subjected to mechanistic
studies using a multifaceted approach consisting of molecular probe design/synthesis, product analysis,
spectroscopic (EPR and Mössbauer) characterization, transient enzyme kinetics, and protein structure prediction
and computational modeling. The acquired mechanistic insights, together with bioinformatics and retro-synthetic
analysis, will be used to guide Fe/2OG enzyme functional (re)assignment in genomic data, to formulate effective
strategies for conversion of hydroxylases to cyclopropanases or aziridinases, and to enable alternative carbo-
and hetero-cycle formations. In the isonitrilase project, we will define factors that govern substrate selectivity of
these enzymes, elucidate product structures, innovate a versatile method to prepare isonitrile-containing
molecules, and assess their metal-binding properties.

## Key facts

- **NIH application ID:** 10803794
- **Project number:** 2R01GM127588-06
- **Recipient organization:** NORTH CAROLINA STATE UNIVERSITY RALEIGH
- **Principal Investigator:** Wei-chen Chang
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $418,445
- **Award type:** 2
- **Project period:** 2018-06-01 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10803794

## Citation

> US National Institutes of Health, RePORTER application 10803794, Understanding How Nonheme Iron-Dependent Enzymes Assemble Pharmacophores: Studies of Cyclopropane, Aziridine, and Isonitrile Formation (2R01GM127588-06). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10803794. Licensed CC0.

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