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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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Manganese binding sites of the oxalate decarboxylase monomer and homology models of the manganese binding sites of CsOxOx.(A) OxDC (PDB ID 1UW8) [32] with manganese ions (purple), metal coordinating residues (atoms colored as follows: C, cyan; N, blue; O, red), conserved active site arginine residues (dark blue) and the N-terminal lid region (green) highlighted. (B) Homology model of the N-terminal CsOxOx Mn binding site metal coordinating residues and the DASN of the lid region. (C) Homology model of the C-terminal CsOxOx Mn binding site metal coordinating residues. The homology model of CsOxOx was constructed using its amino acid sequence and the experimentally solved structure of Bacillus subtilis OxDC (PDB ID 1UW8) using Swiss-Model (The Swiss Institute of Bioinformatics) [48], [49], [50]. Figure generated using Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
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pone-0057933-g003: Manganese binding sites of the oxalate decarboxylase monomer and homology models of the manganese binding sites of CsOxOx.(A) OxDC (PDB ID 1UW8) [32] with manganese ions (purple), metal coordinating residues (atoms colored as follows: C, cyan; N, blue; O, red), conserved active site arginine residues (dark blue) and the N-terminal lid region (green) highlighted. (B) Homology model of the N-terminal CsOxOx Mn binding site metal coordinating residues and the DASN of the lid region. (C) Homology model of the C-terminal CsOxOx Mn binding site metal coordinating residues. The homology model of CsOxOx was constructed using its amino acid sequence and the experimentally solved structure of Bacillus subtilis OxDC (PDB ID 1UW8) using Swiss-Model (The Swiss Institute of Bioinformatics) [48], [49], [50]. Figure generated using Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

Mentions: OxOx activity has been detected in wheat [11], barley [1], [12], [13], beet [14], [15], sorghum [16], [17], maize, oats, rice, and rye [10], [18]. Fungal OxOx activity was first reported in Ceriporiopsis subvermispora, a white rot basidiomycete fungus able to degrade lignin [19]. Plant OxOx enzymes have been structurally characterized and classified as monocupins [5], [20], [21], [22]. In the absence of structural data, prior homology modeling studies predicted that the C. subvermispora enzyme (CsOxOx) is the first manganese-containing bicupin enzyme identified that catalyzes oxalate oxidation [23]. CsOxOx shares greatest sequence identity (49%) with bicupin microbial oxalate decarboxylases (OxDC), which catalyzes the carbon-carbon bond cleavage of oxalate to yield carbon dioxide and formate (Figure 2) [23], [24], [25]. OxDC (and by homology, CsOxOx) is composed of two nearly structurally equivalent β-barrel domains each containing a manganese ion coordinated by four conserved amino acid residues (3 histidine and 1 glutamate) (Figure 3).


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)

Manganese binding sites of the oxalate decarboxylase monomer and homology models of the manganese binding sites of CsOxOx.(A) OxDC (PDB ID 1UW8) [32] with manganese ions (purple), metal coordinating residues (atoms colored as follows: C, cyan; N, blue; O, red), conserved active site arginine residues (dark blue) and the N-terminal lid region (green) highlighted. (B) Homology model of the N-terminal CsOxOx Mn binding site metal coordinating residues and the DASN of the lid region. (C) Homology model of the C-terminal CsOxOx Mn binding site metal coordinating residues. The homology model of CsOxOx was constructed using its amino acid sequence and the experimentally solved structure of Bacillus subtilis OxDC (PDB ID 1UW8) using Swiss-Model (The Swiss Institute of Bioinformatics) [48], [49], [50]. Figure generated using Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585803&req=5

pone-0057933-g003: Manganese binding sites of the oxalate decarboxylase monomer and homology models of the manganese binding sites of CsOxOx.(A) OxDC (PDB ID 1UW8) [32] with manganese ions (purple), metal coordinating residues (atoms colored as follows: C, cyan; N, blue; O, red), conserved active site arginine residues (dark blue) and the N-terminal lid region (green) highlighted. (B) Homology model of the N-terminal CsOxOx Mn binding site metal coordinating residues and the DASN of the lid region. (C) Homology model of the C-terminal CsOxOx Mn binding site metal coordinating residues. The homology model of CsOxOx was constructed using its amino acid sequence and the experimentally solved structure of Bacillus subtilis OxDC (PDB ID 1UW8) using Swiss-Model (The Swiss Institute of Bioinformatics) [48], [49], [50]. Figure generated using Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).
Mentions: OxOx activity has been detected in wheat [11], barley [1], [12], [13], beet [14], [15], sorghum [16], [17], maize, oats, rice, and rye [10], [18]. Fungal OxOx activity was first reported in Ceriporiopsis subvermispora, a white rot basidiomycete fungus able to degrade lignin [19]. Plant OxOx enzymes have been structurally characterized and classified as monocupins [5], [20], [21], [22]. In the absence of structural data, prior homology modeling studies predicted that the C. subvermispora enzyme (CsOxOx) is the first manganese-containing bicupin enzyme identified that catalyzes oxalate oxidation [23]. CsOxOx shares greatest sequence identity (49%) with bicupin microbial oxalate decarboxylases (OxDC), which catalyzes the carbon-carbon bond cleavage of oxalate to yield carbon dioxide and formate (Figure 2) [23], [24], [25]. OxDC (and by homology, CsOxOx) is composed of two nearly structurally equivalent β-barrel domains each containing a manganese ion coordinated by four conserved amino acid residues (3 histidine and 1 glutamate) (Figure 3).

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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