Kinetic and spectroscopic studies of bicupin oxalate oxidase and putative active site mutants
| Autor: | Richard Uberto, Eric Hoffer, Alexander Angerhofer, Bridget Immelman, Morgan Grant, Andrew Ozarowski, Patricia Moussatche, Ellen W. Moomaw, John C. Salerno |
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| Rok vydání: | 2012 |
| Předmět: |
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Molecular Mutant lcsh:Medicine Biochemistry Physical Chemistry Pichia Substrate Specificity chemistry.chemical_compound 0302 clinical medicine Catalytic Domain Aspartic acid lcsh:Science 0303 health sciences Fungal protein Oxalates Multidisciplinary biology Chemistry Physics 030302 biochemistry & molecular biology Hydrogen-Ion Concentration Recombinant Proteins Enzymes Reaction Dynamics Protons Oxidoreductases Biotechnology Binding domain Protein Binding Research Article Stereochemistry Oxalate oxidase Biophysics Arginine Protein Chemistry Oxalate Fungal Proteins Inorganic Chemistry 03 medical and health sciences Chemical Biology Genetics Reaction Kinetics Enzyme kinetics Carboxylate Binding site Molecular Biology Biology Bioinorganic Chemistry 030304 developmental biology Enzyme Kinetics Aspartic Acid Manganese lcsh:R Active site Proteins Kinetics Mutation biology.protein Biocatalysis lcsh:Q Coriolaceae 030217 neurology & neurosurgery |
| Zdroj: | PLoS ONE PLoS ONE, Vol 8, Iss 3, p e57933 (2013) |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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