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Kinetic and spectroscopic studies of bicupin oxalate oxidase and putative active site mutants.

Moomaw EW, Hoffer E, Moussatche P, Salerno JC, Grant M, Immelman B, Uberto R, Ozarowski A, Angerhofer A - PLoS ONE (2013)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, Georgia, United States of America. emoomaw@kennesaw.edu

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.

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CD spectra of recombinant, wild-type CsOxOx and the putative active site mutants.All samples were 938 ug/mL in phosphate buffer (pH 7.0): wild-type CsOxOx, black; D241A, dark blue; D241S, grey; A242E, orange; DASN241-244SENS, red; R169K, cyan; R349K, yellow.
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pone-0057933-g005: CD spectra of recombinant, wild-type CsOxOx and the putative active site mutants.All samples were 938 ug/mL in phosphate buffer (pH 7.0): wild-type CsOxOx, black; D241A, dark blue; D241S, grey; A242E, orange; DASN241-244SENS, red; R169K, cyan; R349K, yellow.

Mentions: Circular dichroism (CD) measurements were carried out to monitor the degree of secondary structural changes due to the site-specific replacement of the putative active site residues under study. The CD spectrum of recombinant, wild type CsOxOx (Figure 5) exhibited a single minimum at 213 nm which is consistent with expectations for a protein composed primarily of β-strands. Three of the six CsOxOx mutants (D241A, A242E, and DASN241-244SENS) showed spectra that closely resemble the spectra of the wild type enzyme with a single minimum at 214, 215, 215 nm, respectively. These data suggest that the global protein folding of the D241A, A242E, and DASN241-244SENS CsOxOx mutants was not disrupted relative to the wild type protein. CsOxOx mutants D241S, R169K, and R349K also displayed a single minimum (220, 220, and 217 nm, respectively) in the region associated with β-strand character but with decreased molar ellipticity values suggesting that these mutations had greater impact on the overall bicupin architecture of these proteins.


Kinetic and spectroscopic studies of bicupin oxalate oxidase and putative active site mutants.

Moomaw EW, Hoffer E, Moussatche P, Salerno JC, Grant M, Immelman B, Uberto R, Ozarowski A, Angerhofer A - PLoS ONE (2013)

CD spectra of recombinant, wild-type CsOxOx and the putative active site mutants.All samples were 938 ug/mL in phosphate buffer (pH 7.0): wild-type CsOxOx, black; D241A, dark blue; D241S, grey; A242E, orange; DASN241-244SENS, red; R169K, cyan; R349K, yellow.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585803&req=5

pone-0057933-g005: CD spectra of recombinant, wild-type CsOxOx and the putative active site mutants.All samples were 938 ug/mL in phosphate buffer (pH 7.0): wild-type CsOxOx, black; D241A, dark blue; D241S, grey; A242E, orange; DASN241-244SENS, red; R169K, cyan; R349K, yellow.
Mentions: Circular dichroism (CD) measurements were carried out to monitor the degree of secondary structural changes due to the site-specific replacement of the putative active site residues under study. The CD spectrum of recombinant, wild type CsOxOx (Figure 5) exhibited a single minimum at 213 nm which is consistent with expectations for a protein composed primarily of β-strands. Three of the six CsOxOx mutants (D241A, A242E, and DASN241-244SENS) showed spectra that closely resemble the spectra of the wild type enzyme with a single minimum at 214, 215, 215 nm, respectively. These data suggest that the global protein folding of the D241A, A242E, and DASN241-244SENS CsOxOx mutants was not disrupted relative to the wild type protein. CsOxOx mutants D241S, R169K, and R349K also displayed a single minimum (220, 220, and 217 nm, respectively) in the region associated with β-strand character but with decreased molar ellipticity values suggesting that these mutations had greater impact on the overall bicupin architecture of these proteins.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, Georgia, United States of America. emoomaw@kennesaw.edu

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.

Show MeSH