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Initial flavin transfer studies on the sulfur-degrading enzyme Dimethyl Sulfide Monooxygenase

Award Number: R03ES026305
ORGANIZATION: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Project Summary/Abstract: The objective of the proposed research is to initiate flavin transfer mechanistic studies on the protein dimethyl sulfide monooxygenase (DMS monooxygenase). DMS monooxygenase catalyzes the conversion of dimethyl sulfide to methanethiol and formaldehyde. DMS monooxygenase is a two-component FMNH2-dependent monooxygenase that requires a DmoA monooxygenase subunit and a DmoB flavin reductase subunit. Both subunits require a flavin mononucleotide (FMN) cofactor for activity. The mechanism surrounding the flavin transfer from DmoA to DmoB remains elusive. Though there are some clues regarding biological function, the specific molecular details surrounding this enzyme mechanism remain unknown.! Initial studies to identify the native DmoB protein of DMS monooxygenase from Hyphomicrobium sulfonivorans will be defined by three approaches. There are two putative flavin reductase proteins located on the dmo gene cluster. Initial kinetic experiments to define the specific cofactors required for activity will be performed, followed by fluorimetric experiments to quantitate flavin binding and stoichiometry. Finally coupled activity assay measurements of the DmoA subunit with the separate DmoB candidates will be performed. Once the native DmoB protein has been determined, flavin transfer mechanism studies will be initiated. The protein- protein interactions studies of DMS monooxygenase will be defined by three approaches. Initial characterization will utilize affinity chromatography by His-tagged DmoA affixed to a Ni-NTA column to identify strong, static protein binding partners. Gel filtration studies will be used similarly to identify strong binding partners. The formation of a stable DmoA:DmoB protein complex will be detected by several analytical techniques including western blot analysis and native PAGE. Finally, fluorescence anisotropy measurements are proposed to characterize the DmoA:DmoB interaction, and will quantitate the binding interaction among the two subunits. This proposal is relevant to the mission of the NIH by developing alternate strategies to mitigate warming trends caused by greenhouse gas emissions. In addition, the results of this proposal will produce a new model system for studying climate change, in particular the enzymatic degradation of volatile organic sulfur compounds (VOSC). Dimethyl sulfide (DMS) is the major contributing biogenic VOSC released into our atmosphere, and is implicated in climate cooling trends. Climate warming has a direct effect on human health by increasing cases of water-born and insect transmitted diseases. Additionally, sulfate aerosol inhalation as a result of VOSC release is linked to pulmonary and heart disease. The results of this work will develop in vitro models to mimic DMS degradation in the environment, its role in climate change and ultimately human health. !


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: 2019 ( Subtotal = $0 )
2019	2018	APPALACHIAN STATE UNIVERSITY	438 ACADEMY ST	BOONE	NC	28607	WATAUGA	USA	Environmental Health	000	2	6/28/2019	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2018 ( Subtotal = $65,663 )
2018	2018	APPALACHIAN STATE UNIVERSITY	1039 STATE FARM RD	BOONE	NC	28607	WATAUGA	USA	Environmental Health	000	2	2/2/2018	NON-COMPETING CONTINUATION	$65,663
														Subtotal = $65,663

Issue Date FY: 2017 ( Subtotal = $62,719 )
2017	2017	APPALACHIAN STATE UNIVERSITY	1039 STATE FARM RD	BOONE	NC	28607	WATAUGA	USA	Environmental Health	000	1	1/31/2017	NEW	$62,719
														Subtotal = $62,719

Grand Total All Awards = $128,382

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Initial flavin transfer studies on the sulfur-degrading enzyme Dimethyl Sulfide Monooxygenase

Award Number: R03ES026305

ORGANIZATION: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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