# Dissect Mechanism of Iron(II)/2-Oxoglutarate Dependent Enzymes Catalyzed Halogenation in Nucleotide Biosynthesis

> **NIH NIH R01** · CARNEGIE-MELLON UNIVERSITY · 2024 · $112,049

## Abstract

Project Summary
Iron and 2-oxoglutarate-dependent (Fe/2OG) enzymes, representing a superfamily of non-heme mononuclear
iron-containing (NHM-Fe) enzymes, have garnered strong research interests from fundamental enzyme
mechanism studies to bioengineering/biocatalysis explorations in recent years due to their exceedingly diverse
catalytic reactivities and simple enzyme architectures. Radical halogenation reactions via C-H bond activation
catalyzed by Fe/2OG halogenases are particularly attractive for chemical synthesis and biocatalysis applications,
since these enzymes can install carbon-halide bonds in a regio- and stereo-specific manner, a feat that has yet
to be achieved by organic synthetic methodology. As revealed by the mechanistic studies of carrier protein
dependent Fe/2OG halogenases, the key step in the radical halogenation mechanism is the selective halide
radical transfer from the hydroxo-Fe(III)-halide intermediate to the substrate radical generated by the key reactive
species, the ferryl (Fe(IV)=O) intermediate. However, a consensus mechanism to explain the selective halide
transfer in Fe/2OG halogenases has not been reached, particularly the controlling factors to avoid hydroxyl
radical transfer to lead to hydroxylation reaction are not fully revealed. Additionally, the reasons why Fe/2OG
enzymes cannot perform fluorination reaction are completely unknown. In this project, we will bridge these
knowledge gaps by studying two newly discovered carrier protein-independent Fe/2OG halogenases that
catalyze chlorination reactions to generate halogenated nucleotide natural products and halogenated
freestanding amino acids. By using an integrative approach consisting of mechanistic probe design and
synthesis, enzyme product structural determination via LC-MS and NMR analysis, transient enzyme kinetics,
advanced spectroscopic characterization and molecular dynamics simulations, we will elucidate the influence of
protein substrate interactions and dynamics in controlling efficient halogenation, explore the effect of different
iron-bound anions (e.g. Cl- vs. F-) to the electronic structure and the reactivity of the ferryl intermediate, test new
chemical strategies to enable fluorination in Fe/2OG enzymes, and expand the substrate scope of these
enzymes for potential synthetic applications. Given the importance of halogen-containing organic molecules in
the modern pharmaceutical and agrochemical applications, mechanistic elucidation of these newly discovered
halogenases will lay scientific foundation for future biocatalytic applications of these unique enzymes.

## Key facts

- **NIH application ID:** 11100111
- **Project number:** 3R01GM125924-06S1
- **Recipient organization:** CARNEGIE-MELLON UNIVERSITY
- **Principal Investigator:** Yisong Guo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $112,049
- **Award type:** 3
- **Project period:** 2018-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11100111

## Citation

> US National Institutes of Health, RePORTER application 11100111, Dissect Mechanism of Iron(II)/2-Oxoglutarate Dependent Enzymes Catalyzed Halogenation in Nucleotide Biosynthesis (3R01GM125924-06S1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/11100111. Licensed CC0.

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