# Probing the Unified Radical Generation Steps in Radical SAM Enzyme Chemistry

> **NIH NIH F32** · MONTANA STATE UNIVERSITY - BOZEMAN · 2021 · $64,926

## Abstract

Project Summary.
 The radical S-adenosyl-L-methionine (SAM) superfamily of enzymes are found in all kingdoms of life and
use an iron-sulfur cluster to catalyze a broad range of reactions. These diverse reactions are surprisingly initiated
by a unified process which begins with the binding of co-substrate/co-factor, SAM, to an iron-sulfur cluster.
Reductive cleavage of SAM by the iron-sulfur cluster generates the reactive radical intermediate, a 5-
deoxyadenosyl radical. This species is a potent hydrogen atom abstractor and is widely accepted as the species
responsible for radical SAM enzymes’ reactivity. The generation of this intermediate has yet to be fully
understood since its presence in the reaction pathway has until recently only been inferred from indirect detection
methods. Building on the recent direct observation of this intermediate using photolysis in the absence of
substrate, as well as through the use non-native substrates, this project aims to capture and characterize the
radical intermediate under catalytically relevant conditions, and to link an organometallic intermediate found to
be central to catalysis to this potent organic radical intermediate. Several approaches will be used to probe the
presence of these intermediates in the reaction pathway resulting in substrate radical generation. A peptide
mimic of the macromolecular substrate of a well-studied radical SAM enzyme will be used to capture the 5-
deoxyadenosyl radical as a stable species. Secondly, another well-studied SAM enzyme whose reaction can be
slowed under particular conditions will allow for the opportunity to study the reactive radical intermediates. These
two enzyme systems will be used with photolysis, rapid freeze quench, cryoreduction, and sequential annealing
techniques. Characterization of intermediates will be carried out using electron paramagnetic resonance and
electron nuclear double resonance spectroscopic experiments, as well as X-ray crystallography. The use of SAM
isotopologues will perturb the spectroscopic signals of potential intermediates, allowing structural identification
of transient intermediates. The objective of the proposed research is to connect proposed catalytic intermediates
to function and provide electronic and geometric details to aid in elucidation of the mechanism of radical initiation
by radical SAM enzymes. Understanding this biochemical reaction will allow for the application of these enzymes
as a powerful biocatalyts capable of numerous challenging reactions and for the potential to pharmaceutically
target radical SAM enzymes in humans and pathogens.
 I aim to launch an independent research career in bioinorganic chemistry. This field is made up of facets
of inorganic chemistry, molecular biology, physics and biochemistry and can have profound impacts on
physiology, pharmacology and toxicology. In order to best be able to contribute to this field, my training must
expand to include aspects of biological sciences. This fell...

## Key facts

- **NIH application ID:** 10155128
- **Project number:** 1F32GM140713-01
- **Recipient organization:** MONTANA STATE UNIVERSITY - BOZEMAN
- **Principal Investigator:** Maike Nicole Lundahl
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $64,926
- **Award type:** 1
- **Project period:** 2021-03-01 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10155128

## Citation

> US National Institutes of Health, RePORTER application 10155128, Probing the Unified Radical Generation Steps in Radical SAM Enzyme Chemistry (1F32GM140713-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10155128. Licensed CC0.

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