# Dissect Mechanism of Iron(II)/2-Oxoglutarate Dependent Enzymes Catalyzed Halogenation in Nucleotide Biosynthesis > **NIH NIH R01** · CARNEGIE-MELLON UNIVERSITY · 2024 · $341,407 ## Abstract Project Summary/Abstract 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 free- standing 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:** 10918144 - **Project number:** 5R01GM125924-07 - **Recipient organization:** CARNEGIE-MELLON UNIVERSITY - **Principal Investigator:** Yisong Guo - **Activity code:** R01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $341,407 - **Award type:** 5 - **Project period:** 2018-09-01 → 2027-08-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10918144 ## Citation > US National Institutes of Health, RePORTER application 10918144, Dissect Mechanism of Iron(II)/2-Oxoglutarate Dependent Enzymes Catalyzed Halogenation in Nucleotide Biosynthesis (5R01GM125924-07). Retrieved via AI Analytics 2026-06-03 from https://api.ai-analytics.org/grant/nih/10918144. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*