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Probing the Unified Radical Generation Steps in Radical SAM Enzyme Chemistry

Award Number: F32GM140713
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 03/01/2021
PERIOD OF PERFORMANCE END DATE: 04/30/2023

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Probing the Unified Radical Generation Steps in Radical SAM Enzyme Chemistry - 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 fellowship in the Broderick laboratory will allow me to gain experience in the expression and purification of proteins and build on experience in elucidation inorganic reaction mechanisms and spectroscopic skills. There is an incredible community of scientists at Montana State University that can expand my understanding of all facets of bioinorganic chemistry.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = -$5,611 )
2024	2023	MONTANA STATE UNIVERSITY	216 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Biomedical Research and Research Training	000	3	2/26/2024	NON-COMPETING CONTINUATION	-$5,611
														Subtotal = -$5,611

Issue Date FY: 2023 ( Subtotal = $16,032 )
2023	2023	MONTANA STATE UNIVERSITY	216 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Biomedical Research and Research Training	000	3	2/9/2023	NON-COMPETING CONTINUATION	$71,792
2023	2023	MONTANA STATE UNIVERSITY	216 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Biomedical Research and Research Training	001	3	5/30/2023	NON-COMPETING CONTINUATION	-$55,760
														Subtotal = $16,032

Issue Date FY: 2022 ( Subtotal = $67,582 )
2022	2022	MONTANA STATE UNIVERSITY	216 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Biomedical Research and Research Training	001	2	5/11/2022	NON-COMPETING CONTINUATION	$1,191
2022	2022	MONTANA STATE UNIVERSITY	216 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Biomedical Research and Research Training	000	2	2/11/2022	NON-COMPETING CONTINUATION	$66,390
2022	2022	MONTANA STATE UNIVERSITY	216 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Biomedical Research and Research Training	002	2	6/22/2022	NON-COMPETING CONTINUATION	$1
														Subtotal = $67,582

Issue Date FY: 2021 ( Subtotal = $64,926 )
2021	2021	MONTANA STATE UNIVERSITY, INC	211 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Biomedical Research and Research Training	002	1	2/4/2021	NEW	$0
2021	2021	MONTANA STATE UNIVERSITY, INC	211 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Biomedical Research and Research Training	001	1	2/4/2021	NEW	$0
2021	2021	MONTANA STATE UNIVERSITY, INC	211 MONTANA HALL	BOZEMAN	MT	59717	GALLATIN	USA	Biomedical Research and Research Training	000	1	2/4/2021	NEW	$64,926
														Subtotal = $64,926

Grand Total All Awards = $142,929

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Probing the Unified Radical Generation Steps in Radical SAM Enzyme Chemistry

Award Number: F32GM140713

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 03/01/2021

PERIOD OF PERFORMANCE END DATE: 04/30/2023

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