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Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzy...

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Autores principales: Moomaw, Ellen W., Hoffer, Eric, Moussatche, Patricia, Salerno, John C., Grant, Morgan, Immelman, Bridget, Uberto, Richard, Ozarowski, Andrew, Angerhofer, Alexander
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585803/
https://www.ncbi.nlm.nih.gov/pubmed/23469254
http://dx.doi.org/10.1371/journal.pone.0057933
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author Moomaw, Ellen W.
Hoffer, Eric
Moussatche, Patricia
Salerno, John C.
Grant, Morgan
Immelman, Bridget
Uberto, Richard
Ozarowski, Andrew
Angerhofer, Alexander
author_facet Moomaw, Ellen W.
Hoffer, Eric
Moussatche, Patricia
Salerno, John C.
Grant, Morgan
Immelman, Bridget
Uberto, Richard
Ozarowski, Andrew
Angerhofer, Alexander
author_sort Moomaw, Ellen W.
collection PubMed
description Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.
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spelling pubmed-35858032013-03-06 Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants Moomaw, Ellen W. Hoffer, Eric Moussatche, Patricia Salerno, John C. Grant, Morgan Immelman, Bridget Uberto, Richard Ozarowski, Andrew Angerhofer, Alexander PLoS One Research Article Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. Public Library of Science 2013-03-01 /pmc/articles/PMC3585803/ /pubmed/23469254 http://dx.doi.org/10.1371/journal.pone.0057933 Text en © 2013 Moomaw et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Moomaw, Ellen W.
Hoffer, Eric
Moussatche, Patricia
Salerno, John C.
Grant, Morgan
Immelman, Bridget
Uberto, Richard
Ozarowski, Andrew
Angerhofer, Alexander
Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants
title Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants
title_full Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants
title_fullStr Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants
title_full_unstemmed Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants
title_short Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants
title_sort kinetic and spectroscopic studies of bicupin oxalate oxidase and putative active site mutants
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585803/
https://www.ncbi.nlm.nih.gov/pubmed/23469254
http://dx.doi.org/10.1371/journal.pone.0057933
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