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NADP(H)-dependent biocatalysis without adding NADP(H)

Isocitrate dehydrogenase 1 (IDH1) naturally copurifies and crystallizes in a resting state with a molecule of its exchangeable cofactor, NADP(+)/NADPH, bound in each monomer of the homodimer. We report electrochemical studies with IDH1 that exploit this property to reveal the massive advantage of na...

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Autores principales: Herold, Ryan A., Reinbold, Raphael, Schofield, Christopher J., Armstrong, Fraser A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9910440/
https://www.ncbi.nlm.nih.gov/pubmed/36574703
http://dx.doi.org/10.1073/pnas.2214123120
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author Herold, Ryan A.
Reinbold, Raphael
Schofield, Christopher J.
Armstrong, Fraser A.
author_facet Herold, Ryan A.
Reinbold, Raphael
Schofield, Christopher J.
Armstrong, Fraser A.
author_sort Herold, Ryan A.
collection PubMed
description Isocitrate dehydrogenase 1 (IDH1) naturally copurifies and crystallizes in a resting state with a molecule of its exchangeable cofactor, NADP(+)/NADPH, bound in each monomer of the homodimer. We report electrochemical studies with IDH1 that exploit this property to reveal the massive advantage of nanoconfinement to increase the efficiency of multistep enzyme-catalyzed cascade reactions. When coloaded with ferredoxin NADP(+) reductase in a nanoporous conducting indium tin oxide film, IDH1 carries out the complete electrochemical oxidation of 6 mM isocitrate (in 4mL) to 2-oxoglutarate (2OG), using only the NADP(H) that copurified with IDH1 and was carried into the electrode pores as cargo—the system remains active for days. The entrapped cofactor, now quantifiable by cyclic voltammetry, undergoes ~160,000 turnovers during the process. The results from a variety of electrocatalysis experiments imply that the local concentrations of the two nanoconfined enzymes lie around the millimolar range. The combination of crowding and entrapment results in a 10(2) to 10(3)-fold increase in the efficiency of NADP(H) redox cycling. The ability of the method to drive cascade catalysis in either direction (oxidation or reduction) and remove and replace substrates was exploited to study redox-state dependent differences in cofactor binding between wild-type IDH1 and the cancer-linked R132H variant that catalyzes the “gain of function” reduction of 2OG to 2-hydroxyglutarate instead of isocitrate oxidation. The combined results demonstrate the power of nanoconfinement for facilitating multistep enzyme catalysis (in this case energized and verified electrochemically) and reveal insights into the dynamic role of nicotinamide cofactors as redox (hydride) carriers.
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spelling pubmed-99104402023-02-10 NADP(H)-dependent biocatalysis without adding NADP(H) Herold, Ryan A. Reinbold, Raphael Schofield, Christopher J. Armstrong, Fraser A. Proc Natl Acad Sci U S A Biological Sciences Isocitrate dehydrogenase 1 (IDH1) naturally copurifies and crystallizes in a resting state with a molecule of its exchangeable cofactor, NADP(+)/NADPH, bound in each monomer of the homodimer. We report electrochemical studies with IDH1 that exploit this property to reveal the massive advantage of nanoconfinement to increase the efficiency of multistep enzyme-catalyzed cascade reactions. When coloaded with ferredoxin NADP(+) reductase in a nanoporous conducting indium tin oxide film, IDH1 carries out the complete electrochemical oxidation of 6 mM isocitrate (in 4mL) to 2-oxoglutarate (2OG), using only the NADP(H) that copurified with IDH1 and was carried into the electrode pores as cargo—the system remains active for days. The entrapped cofactor, now quantifiable by cyclic voltammetry, undergoes ~160,000 turnovers during the process. The results from a variety of electrocatalysis experiments imply that the local concentrations of the two nanoconfined enzymes lie around the millimolar range. The combination of crowding and entrapment results in a 10(2) to 10(3)-fold increase in the efficiency of NADP(H) redox cycling. The ability of the method to drive cascade catalysis in either direction (oxidation or reduction) and remove and replace substrates was exploited to study redox-state dependent differences in cofactor binding between wild-type IDH1 and the cancer-linked R132H variant that catalyzes the “gain of function” reduction of 2OG to 2-hydroxyglutarate instead of isocitrate oxidation. The combined results demonstrate the power of nanoconfinement for facilitating multistep enzyme catalysis (in this case energized and verified electrochemically) and reveal insights into the dynamic role of nicotinamide cofactors as redox (hydride) carriers. National Academy of Sciences 2022-12-27 2023-01-03 /pmc/articles/PMC9910440/ /pubmed/36574703 http://dx.doi.org/10.1073/pnas.2214123120 Text en Copyright © 2022 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by/4.0/This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Biological Sciences
Herold, Ryan A.
Reinbold, Raphael
Schofield, Christopher J.
Armstrong, Fraser A.
NADP(H)-dependent biocatalysis without adding NADP(H)
title NADP(H)-dependent biocatalysis without adding NADP(H)
title_full NADP(H)-dependent biocatalysis without adding NADP(H)
title_fullStr NADP(H)-dependent biocatalysis without adding NADP(H)
title_full_unstemmed NADP(H)-dependent biocatalysis without adding NADP(H)
title_short NADP(H)-dependent biocatalysis without adding NADP(H)
title_sort nadp(h)-dependent biocatalysis without adding nadp(h)
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9910440/
https://www.ncbi.nlm.nih.gov/pubmed/36574703
http://dx.doi.org/10.1073/pnas.2214123120
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