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Mechanisms of novel biological nitrogen chemistries

Award Number: R35GM147515
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/01/2022
PERIOD OF PERFORMANCE END DATE: 07/31/2027

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Mechanisms of novel biological nitrogen chemistries - The proposed research will study the mechanisms and structure-function relationships of novel nitric oxide (NO)-dependent enzymatic activities with relevance to protein nitration, natural product biosynthesis, and nitrosative stress protection in pathogenic bacteria. Exposure of cells to NO results in cell damage including nitration of protein residues such as tyrosine (Tyr) and tryptophan (Trp). Protein nitration can modulate protein function and are biomarkers of some neurodegenerative diseases. Some protein nitration can be catalyzed by metal ions. The mechanisms of metal-mediated nitration pathways are unclear; therefore, Research Direction 1 is to study the mechanisms of metal-catalyzed protein nitration. To better understand these mechanisms, we will study the nitration by cytochromes P450 (CYPs), including that of the enzyme TxtE, a CYP homolog that catalyzes the regiospecific and NO-dependent nitration of Trp to 4-nitrotryptophan (4-NO2-Trp). The observed regiospecificity suggests that diffusible RNS are not produced on pathway. Furthermore, CYP metal-oxo intermediates have been well characterized. Therefore, studies of TxtE have great potential to provide clear mechanistic data on nitration. One challenge is that TxtE nitration intermediates are elusive. Therefore, the 5-year goal of Research Direction 1 is to determine the outer sphere coordination features of TxtE needed to promote substrate nitration and avoid substrate hydroxylation, the latter being canonical CYP activity. Identifying outer sphere interactions that influence TxtE reactivity will provide structure-function insight to devise strategies to trap nitration intermediates. To avoid cell damage from NO, pathogenic Mycobacteria express enzymes to scavenge NO. Research Direction 2 will study activities and mechanisms of metalloenzymes involved in Mycobacterial virulence, including hemerythrin-like proteins (HLPs) found in pathogenic Mycobacteria, including M. tuberculosis. Our preliminary data on HLPs shows that its diferric oxidation state can oxidize NO to nitrite (NO2–) by reductive nitrosylation. Such reactivity is well known for heme enzymes but has never been reported for a non-heme protein. In the presence of O2, HLP reacts with NO to form nitrate (NO3–) and a metalloproduct with a 350-nm absorbance feature. This metalloproduct can be independently generated by reacting the as isolated HLP with peroxynitrite (ONOO–). The novel reactivities of HLP with RNS suggest a role for it in nitrosative stress protection. An uncommon Tyr ligand bound to the HLP non-heme diiron site may facilitate these unusual reactivities. The 5-year goals of Research Direction 2 are to trap and characterize intermediates of RNS reactions with HLP, determine the catalytic activity of HLP, and identify the role of the Tyr ligand. The vision of our program is to discover novel nitrogen biochemistries related to natural product biosynthesis and human health and to pursue engineering biocatalysts and to establish a world-class training program in mechanistic metalloenzymology and protein engineering, and natural product biosynthesis at UCF.


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: 2025 ( Subtotal = $364,638 )
2025	2025	THE UNIVERSITY OF CENTRAL FLORIDA BOARD OF TRUSTEES	4000 CENTRAL FLORIDA BLVD	ORLANDO	FL	32816	ORANGE	USA	Biomedical Research and Research Training	000	4	7/28/2025	NON-COMPETING CONTINUATION	$364,638
														Subtotal = $364,638

Issue Date FY: 2024 ( Subtotal = $364,638 )
2024	2024	THE UNIVERSITY OF CENTRAL FLORIDA BOARD OF TRUSTEES	4000 CENTRAL FLORIDA BLVD	ORLANDO	FL	32816	ORANGE	USA	Biomedical Research and Research Training	000	3	7/25/2024	NON-COMPETING CONTINUATION	$364,638
														Subtotal = $364,638

Issue Date FY: 2023 ( Subtotal = $364,638 )
2023	2023	UNIVERSITY OF CENTRAL FLORIDA BOARD OF TRUSTEES, THE	4000 CENTRAL FLORIDA BLVD	ORLANDO	FL	32816	ORANGE	USA	Biomedical Research and Research Training	000	2	6/27/2023	NON-COMPETING CONTINUATION	$364,638
														Subtotal = $364,638

Issue Date FY: 2022 ( Subtotal = $364,638 )
2022	2022	UNIVERSITY OF CENTRAL FLORIDA BOARD OF TRUSTEES, THE	4000 CENTRAL FLORIDA BLVD	ORLANDO	FL	32816	ORANGE	USA	Biomedical Research and Research Training	000	1	7/19/2022	NEW	$364,638
														Subtotal = $364,638

Grand Total All Awards = $1,458,552

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Mechanisms of novel biological nitrogen chemistries

Award Number: R35GM147515

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 08/01/2022

PERIOD OF PERFORMANCE END DATE: 07/31/2027

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