Saturday, November 23, 2024 11/23/2024

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

Award Number: R01GM125924
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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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.


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 = $453,456 )
2024	2024	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	001	6	9/4/2024	SUPPLEMENT FOR EXPANSION	$112,049
2024	2024	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	000	7	8/14/2024	NON-COMPETING CONTINUATION	$341,407
														Subtotal = $453,456

Issue Date FY: 2023 ( Subtotal = $355,071 )
2023	2023	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	000	6	9/1/2023	COMPETING CONTINUATION	$355,071
														Subtotal = $355,071

Issue Date FY: 2022 ( Subtotal = $336,339 )
2022	2022	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	001	5	8/12/2022	SUPPLEMENT FOR EXPANSION	$27,915
2022	2022	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	000	5	8/11/2022	NON-COMPETING CONTINUATION	$308,424
														Subtotal = $336,339

Issue Date FY: 2021 ( Subtotal = $504,512 )
2021	2021	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	002	4	8/11/2021	SUPPLEMENT FOR EXPANSION	$111,652
2021	2021	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	001	4	8/10/2021	NON-COMPETING CONTINUATION	$309,120
2021	2021	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	000	3	11/16/2020	SUPPLEMENT FOR EXPANSION	$83,740
														Subtotal = $504,512

Issue Date FY: 2020 ( Subtotal = $314,201 )
2020	2020	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	000	3	8/7/2020	NON-COMPETING CONTINUATION	$314,201
														Subtotal = $314,201

Issue Date FY: 2019 ( Subtotal = $310,513 )
2019	2019	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	000	2	8/5/2019	NON-COMPETING CONTINUATION	$310,513
														Subtotal = $310,513

Issue Date FY: 2018 ( Subtotal = $328,368 )
2018	2018	CARNEGIE MELLON UNIVERSITY	5000 FORBES AVE	PITTSBURGH	PA	15213	ALLEGHENY	USA	Biomedical Research and Research Training	000	1	7/31/2018	NEW	$328,368
														Subtotal = $328,368

Grand Total All Awards = $2,602,460

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

Award Number: R01GM125924

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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