title: Kinetic and spectroscopic studies of bicupin oxalate oxidase and putative active site mutants.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, Georgia, United States of America. emoomaw@kennesaw.edu
authors.forename: Ellen W
authors.initials: EW
authors.lastname: Moomaw
authors.forename: Eric
authors.initials: E
authors.lastname: Hoffer
authors.forename: Patricia
authors.initials: P
authors.lastname: Moussatche
authors.forename: John C
authors.initials: JC
authors.lastname: Salerno
authors.forename: Morgan
authors.initials: M
authors.lastname: Grant
authors.forename: Bridget
authors.initials: B
authors.lastname: Immelman
authors.forename: Richard
authors.initials: R
authors.lastname: Uberto
authors.forename: Andrew
authors.initials: A
authors.lastname: Ozarowski
authors.forename: Alexander
authors.initials: A
authors.lastname: Angerhofer
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D055453:Coriolaceae / Q000737:chemistry / Q000201:enzymology*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D010070:Oxalates / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D010070:Oxalates; D011522:Protons; D011994:Recombinant Proteins; D001224:Aspartic Acid; D008345:Manganese; D001120:Arginine; D010088:Oxidoreductases; C020598:oxalate oxidase