Citation
Effect Of Dioxygen Exposure On Hydrogen Peroxide Production By Bacillus Subtilis Oxalate Decarboxylase In The Absence Of Substrate

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Title:
Effect Of Dioxygen Exposure On Hydrogen Peroxide Production By Bacillus Subtilis Oxalate Decarboxylase In The Absence Of Substrate
Series Title:
19th Annual Undergraduate Research Symposium
Creator:
Booth, Megan
Language:
English
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Undetermined

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Subjects / Keywords:
Center for Undergraduate Research
Center for Undergraduate Research
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Conference papers and proceedings
Poster

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Abstract:
Oxalate decarboxylase is a manganese dependent enzyme that catalyzes the breakdown of oxalate into carbon dioxide and formate using dioxygen as a cofactor. The role of dioxygen in the enzyme’s mechanism is still under investigation, but it is proposed to prepare the enzyme for catalysis by facilitating Mn oxidation at low pH. In doing so, dioxygen is expected to be reduced to superoxide, which undergoes acid-catalyzed dismutation to form hydrogen peroxide. This hypothesis is supported by new evidence that the concentration of hydrogen peroxide produced by resting enzyme at low pH is significantly higher in dioxygen saturated conditions than in dioxygen limited conditions. ( en )
General Note:
Research authors: Megan Booth and Alexander Angerhofer - University of Florida
General Note:
University Scholars Program
General Note:
Faculty Mentor: Oxalate decarboxylase is a manganese dependent enzyme that catalyzes the breakdown of oxalate into carbon dioxide and formate using dioxygen as a cofactor. The role of dioxygen in the enzyme’s mechanism is still under investigation, but it is proposed to prepare the enzyme for catalysis by facilitating Mn oxidation at low pH. In doing so, dioxygen is expected to be reduced to superoxide, which undergoes acid-catalyzed dismutation to form hydrogen peroxide. This hypothesis is supported by new evidence that the concentration of hydrogen peroxide produced by resting enzyme at low pH is significantly higher in dioxygen saturated conditions than in dioxygen limited conditions. - Center for Undergraduate Research, University Scholars Program

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University of Florida
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Copyright Megan Booth. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Effect of Dioxygen Exposure on Hydrogen Peroxide Production by Bacillus subtilis Oxalate Decarboxylase in the Absence of Substrate Megan Booth and Dr. Alexander Angerhofer Methodology Results Department of Chemistry, University of Florida, Gainesville FL 32611 7200, USA References Oxalate decarboxylase ( OxDC ) from Bacillus subtilis is a manganese dependent enzyme that catalyzes the breakdown of oxalate into CO 2 and formate using bound O 2 as a cofactor. 1,2 It is known that Mn(III) drives the reaction; however, the curious dual dependence on O 2 has garnered attention, including questions such as Where does O 2 bind? Is there a LRET process involved between bound O 2 and the catalytically active Mn? Is O 2 only required to generate Mn(III) or does it actively participate in decarboxylation? The answer to this first question awaits further study; however, new evidence presented herein supports the theory that O 2 binding drives Mn oxidation, acting as an electron sink to form superoxide and its acid catalyzed dismutation product H 2 O 2 Enzyme expression, purification, and concentration Anaerobic dialysis from pH 8.5 to 4.0 and reintroduction of O 2 HyPerBlu chemiluminescent assay to determine extent of H 2 O 2 production Conclusions Introduction Unpublished crystal structure of wild type OxDC Manganese centers are shown in purple. Monomer is denoted in blue. Dioxygen Limited Dioxygen Saturated Final [ OxDC ] 390 30 M 340 30 M [H 2 O 2 ] Produced 2.18 0.07 M 3.22 0.45 M [H 2 O 2 ]/[ OxDC ] 5.6 0.5 (x10 3 ) 9.5 1.5 (x10 3 ) % Difference in [H 2 O 2 ]/[ OxDC ] 70% A 70% increase in H 2 O 2 production was observed upon saturating OxDC with O 2 suggesting a direct correlation between O 2 binding and Mn oxidation Future experiments will be focused on establishing a true baseline for H 2 O 2 by ensuring O 2 is eliminated from the system. This will allow further studies to see if activity is solely dependent on Mn oxidation or if O 2 plays an active role in addition to facilitating oxidation 1. Tanner, A. et al. (2001) J. Biol. Chem. 276 : 2. Twahir U et al. (2016) Biochemistry. 55 : 6506 6516. Funding Funding for this project was provided by the Program