Oxalate oxidase

 

Oxalate oxidase (OXO) catalyses the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. The enzyme is widespread in nature but is most abundant in higher plant tissues, particularly germinating seeds. It is a member of a functionally diverse superfamily known at the cupins or double stranded β-helix proteins containing a mononuclear manganese centre in each subunit.

The mechanism for OXO remains unclear. It is closely related to the bicupin oxalate decarboxylase which also contains manganese but produces carbon dioxide and formate.

 

Reference Protein and Structure

Sequence
P45850 UniProt (1.2.3.4) IPR001929 (Sequence Homologues) (PDB Homologues)
Biological species
Hordeum vulgare (Barley) Uniprot
PDB
2et1 - Oxalate oxidase in complex with substrate analogue glycolate (1.6 Å) PDBe PDBsum 2et1
Catalytic CATH Domains
2.60.120.10 CATHdb (see all for 2et1)
Cofactors
Manganese(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:1.2.3.4)

hydron
CHEBI:15378ChEBI
+
dioxygen
CHEBI:15379ChEBI
+
oxalate(2-)
CHEBI:30623ChEBI
→
hydrogen peroxide
CHEBI:16240ChEBI
+
carbon dioxide
CHEBI:16526ChEBI
Alternative enzyme names: Aero-oxalo dehydrogenase, Oxalic acid oxidase,

Enzyme Mechanism

Introduction

This mechanism begins with octahedral coordination of the manganese ion at the catalytic centre of the enzyme by three histidine residues (His88, His90 and His137), one glutamate (Glu95) and two solvent water molecules. Monodentate coordination of the oxalate substrate displaces a water molecule from the Mn(II) centre. Then dioxygen binding to Mn(II) displaces another solvent molecule, and forms an Mn(III) superoxide metalloradical complex. The superoxide radical performs a nucleophilic attack on the proximal carboxyl group, activating the substrate. Hydrogen atom transfer forms a distal carboxyl radical species, and the substrate rearranges by homolytic C-C bond cleavage and reduction of Mn(III). Lastly, it is possible that release of products includes hydrolysis of percarbonate (not shown).

Catalytic Residues Roles

UniProt PDB* (2et1)
Asn75 Asn75A Asn75 shows conformational flexibility, suggesting it has a dynamic role in catalysis e.g. assisting substrates and products through the access channel. Mutation to alanine results in complete loss of activity. hydrogen bond acceptor
Asn85 Asn85A Along with Gln139, Asn85 facilitates formation of the percarbonate product by ensuring the planarity of the manganese ion and atoms of the dioxygen superoxide and the carbon dioxide fragment from oxalate. Mutation to alanine results in complete loss of activity. hydrogen bond donor
Gln139 Gln139A A manganese ion, together with Asn75, Asn85 and Gln139 comprise a redox active metal cofactor and hydrogen bonding framework important in the orientation and activation of substrates. hydrogen bond acceptor, hydrogen bond donor
His90, His88, His137, Glu95 His90A, His88A, His137A, Glu95A There is octahedral coordination around the manganese ion by His88, His90, Glu95, His137 and two water molecules. metal ligand
Met149, Val77 Met149A, Val77A Leu77 and Met149 provide steric constraints on bidentate coordination in OXO. steric role
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

cofactor used, coordination to a metal ion, decoordination from a metal ion, substitution (not covered by the Ingold mechanisms), radical propagation, colligation, intermediate formation, bimolecular nucleophilic addition, overall reactant used, hydrogen transfer, homolysis, radical termination, intermediate collapse, decarboxylation, overall product formed, native state of cofactor regenerated, inferred reaction step, intramolecular elimination

References

  1. Opaleye O et al. (2006), J Biol Chem, 281, 6428-6433. Structural and spectroscopic studies shed light on the mechanism of oxalate oxidase. DOI:10.1074/jbc.M510256200. PMID:16291738.
  2. Goodwin JM et al. (2017), PLoS One, 12, e0177164-. Hydrogen peroxide inhibition of bicupin oxalate oxidase. DOI:10.1371/journal.pone.0177164. PMID:28486485.
  3. Woo EJ et al. (2000), Nat Struct Biol, 7, 1036-1040. Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities. DOI:10.1038/80954. PMID:11062559.

Step 1. Monodentate coordination of oxalate monoanion, displacing one solvent molecule from the Mn(II) centre. This forms the dioxygen binding site and lowers the redox potential of the Mn(II)-Mn(III) couple, activating the metal ion to bind dioxygen as a superoxide anion. Leu77 and Met149 provide steric constraints on bidentate coordination.

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Catalytic Residues Roles

Residue Roles
Asn85A hydrogen bond donor
Gln139A hydrogen bond donor
Asn75A hydrogen bond acceptor
Gln139A hydrogen bond acceptor
His90A metal ligand
His88A metal ligand
His137A metal ligand
Glu95A metal ligand
Met149A steric role
Val77A steric role

Chemical Components

cofactor used, coordination to a metal ion, decoordination from a metal ion

Step 2. Dioxygen binding to Mn(II) displaces another solvent molecule, and forms an Mn(III) superoxide metalloradical complex.

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Catalytic Residues Roles

Residue Roles
His90A metal ligand
His88A metal ligand
His137A metal ligand
Glu95A metal ligand
Asn85A hydrogen bond donor
Gln139A hydrogen bond donor
Asn75A hydrogen bond acceptor
Gln139A hydrogen bond acceptor

Chemical Components

substitution (not covered by the Ingold mechanisms), coordination to a metal ion, decoordination from a metal ion, cofactor used, radical propagation, colligation, intermediate formation

Step 3. The superoxide radical performs a nucleophilic attack on the proximal carboxyl group, activating the substrate.

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Catalytic Residues Roles

Residue Roles
His90A metal ligand
His88A metal ligand
His137A metal ligand
Glu95A metal ligand
Asn75A hydrogen bond acceptor
Gln139A hydrogen bond acceptor
Asn85A hydrogen bond donor
Gln139A hydrogen bond donor

Chemical Components

ingold: bimolecular nucleophilic addition, cofactor used, intermediate formation, radical propagation, overall reactant used

Step 4. Hydrogen atom transfer forms a distal carboxyl radical species.

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Catalytic Residues Roles

Residue Roles
Asn75A hydrogen bond acceptor
Gln139A hydrogen bond acceptor
Asn85A hydrogen bond donor
Gln139A hydrogen bond donor
His90A metal ligand
His88A metal ligand
His137A metal ligand
Glu95A metal ligand

Chemical Components

hydrogen transfer, radical propagation

Step 5. Substrate radical rearranges with homolytic C-C bond cleavage and reduction of Mn(III) to Mn(II).

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Catalytic Residues Roles

Residue Roles
Asn85A hydrogen bond donor
Gln139A hydrogen bond donor
Asn75A hydrogen bond acceptor
Gln139A hydrogen bond acceptor
His90A metal ligand
His88A metal ligand
His137A metal ligand
Glu95A metal ligand

Chemical Components

homolysis, radical termination, intermediate collapse, decarboxylation, decoordination from a metal ion

Step 6. The remaining products are formed by an intramolecular elimination. Two water molecules bind the metal to regenerate the initial step. This step is inferred by the curator.

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Catalytic Residues Roles

Residue Roles
Asn75A hydrogen bond acceptor
Gln139A hydrogen bond acceptor
Asn85A hydrogen bond donor
Gln139A hydrogen bond donor
His90A metal ligand
His88A metal ligand
His137A metal ligand
Glu95A metal ligand

Chemical Components

overall product formed, native state of cofactor regenerated, inferred reaction step, ingold: intramolecular elimination
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Step 1. Monodentate coordination of oxalate monoanion, displacing one solvent molecule from the Mn(II) centre. This forms the dioxygen binding site and lowers the redox potential of the Mn(II)-Mn(III) couple, activating the metal ion to bind dioxygen as a superoxide anion. Leu77 and Met149 provide steric constraints on bidentate coordination.

Step 2. Dioxygen binding to Mn(II) displaces another solvent molecule, and forms an Mn(III) superoxide metalloradical complex.

Step 3. The superoxide radical performs a nucleophilic attack on the proximal carboxyl group, activating the substrate.

Step 4. Hydrogen atom transfer forms a distal carboxyl radical species.

Step 5. Substrate radical rearranges with homolytic C-C bond cleavage and reduction of Mn(III) to Mn(II).

Step 6. The remaining products are formed by an intramolecular elimination. Two water molecules bind the metal to regenerate the initial step. This step is inferred by the curator.

Products.

Introduction

This mechanism identifies the fraction of the enzyme containing Mn(III) as the active form of the enzyme. Monodentate coordination of the oxalate monoanion to an Mn(III) centre. Under anaerobic conditions, oxalate reduces Mn(III) to Mn(II) and forms an oxalyl free radical. Decarboxylation leaves a carbon dioxide radical anion bound to Mn(II). Dioxygen intercepts the carbon dioxide radical anion, generating a second molecule of carbon dioxide and superoxide. Subsequent electron transfer from Mn(II) to a hydroperoxyl radical generates hydrogen peroxide. This mechanism may be specific to barley oxalate oxidase.

Catalytic Residues Roles

UniProt PDB* (2et1)
Asn85 Asn85A Along with Gln139, Asn85 facilitates formation of the percarbonate product by ensuring the planarity of the manganese ion and atoms of the dioxygen superoxide and the carbon dioxide fragment from oxalate. Mutation to alanine results in complete loss of activity. hydrogen bond donor
Asn75, Asn85, Gln139 Asn75A, Asn85A, Gln139A A manganese ion, together with Asn75, Asn85 and Gln139 comprise a redox active metal cofactor and hydrogen bonding framework important in the orientation and activation of substrates. hydrogen bond acceptor
His90, His88, His137, Glu95 His90A, His88A, His137A, Glu95A There is octahedral coordination around the manganese ion by His88, His90, Glu95, His137 and two water molecules. metal ligand
Met149, Val77 Met149A, Val77A Leu77 and Met149 provide steric constraints on bidentate coordination in OXO. steric role
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

substitution (not covered by the Ingold mechanisms), decoordination from a metal ion, coordination to a metal ion, cofactor used, intermediate formation, proton transfer, decarboxylation, intermediate collapse, electron transfer, overall product formed, radical termination, native state of cofactor regenerated

References

  1. Whittaker MM et al. (2007), J Biol Chem, 282, 7011-7023. Burst kinetics and redox transformations of the active site manganese ion in oxalate oxidase: implications for the catalytic mechanism. DOI:10.1074/jbc.M609374200. PMID:17210574.

Step 1. Monodentate coordination of oxalate monoanion, displacing one solvent molecule from an Mn(III) centre.

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Catalytic Residues Roles

Residue Roles
Val77A steric role
Met149A steric role
Glu95A metal ligand
His137A metal ligand
His88A metal ligand
His90A metal ligand
Gln139A hydrogen bond acceptor
Asn75A hydrogen bond acceptor
Gln139A hydrogen bond donor
Asn85A hydrogen bond donor

Chemical Components

substitution (not covered by the Ingold mechanisms), decoordination from a metal ion, coordination to a metal ion, cofactor used

Step 2. Under anaerobic conditions, oxalate reduces Mn(III) to Mn(II) and forms an oxalyl free radical. Proton transfer inferred by curator.

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Catalytic Residues Roles

Residue Roles
Gln139A hydrogen bond acceptor
Asn75A hydrogen bond acceptor
Gln139A hydrogen bond donor
Asn85A hydrogen bond donor
Glu95A metal ligand
His137A metal ligand
His88A metal ligand
His90A metal ligand

Chemical Components

intermediate formation, cofactor used, decoordination from a metal ion, coordination to a metal ion, substitution (not covered by the Ingold mechanisms), proton transfer

Step 3. Decarboxylation in aqueous conditions releases carbon dioxide and leaves a carbon dioxide radical anion bound to Mn(II).

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Catalytic Residues Roles

Residue Roles

Chemical Components

decarboxylation, intermediate collapse, decoordination from a metal ion

Step 4. Dioxygen intercepts the carbon dioxide radical anion, generating a second molecule of carbon dioxide and superoxide. Protonation inferred by curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

decarboxylation, decoordination from a metal ion, electron transfer

Step 5. Electron transfer from Mn(II) to a hydroperoxyl radical, generating hydrogen peroxide. A other molecule binds the metal ion to regenerate the active site.

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Catalytic Residues Roles

Residue Roles

Chemical Components

electron transfer, overall product formed, proton transfer, radical termination, native state of cofactor regenerated
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Step 1. Monodentate coordination of oxalate monoanion, displacing one solvent molecule from an Mn(III) centre.

Step 2. Under anaerobic conditions, oxalate reduces Mn(III) to Mn(II) and forms an oxalyl free radical. Proton transfer inferred by curator.

Step 3. Decarboxylation in aqueous conditions releases carbon dioxide and leaves a carbon dioxide radical anion bound to Mn(II).

Step 4. Dioxygen intercepts the carbon dioxide radical anion, generating a second molecule of carbon dioxide and superoxide. Protonation inferred by curator.

Step 5. Electron transfer from Mn(II) to a hydroperoxyl radical, generating hydrogen peroxide. A other molecule binds the metal ion to regenerate the active site.

Products.

Introduction

This mechanism is consistent with the results from a study demonstrating that oxalate oxidase and oxalate decarboxylase activities can be interchanged by mutating an active site lid. Oxalate binds to Mn(II), which then undergoes oxidation to Mn(III) upon binding of dioxygen. Reduction of Mn(III) to Mn(II) and decarboxylation occur, which is followed by insertion of the superoxide radical into oxalate. If the insertion occurred before decarboxylation, the mechanism would be committed to oxidation.

Catalytic Residues Roles

UniProt PDB* (2et1)
Asn85 Asn85A Along with Gln139, Asn85 facilitates formation of the percarbonate product by ensuring the planarity of the manganese ion and atoms of the dioxygen superoxide and the carbon dioxide fragment from oxalate. Mutation to alanine results in complete loss of activity. hydrogen bond donor
Asn75, Asn85, Gln139 Asn75A, Asn85A, Gln139A A manganese ion, together with Asn75, Asn85 and Gln139 comprise a redox active metal cofactor and hydrogen bonding framework important in the orientation and activation of substrates. hydrogen bond acceptor
His90, His88, His137, Glu95 His90A, His88A, His137A, Glu95A There is octahedral coordination around the manganese ion by His88, His90, Glu95, His137 and two water molecules. metal ligand
Met149, Val77 Met149A, Val77A Leu77 and Met149 provide steric constraints on bidentate coordination in OXO. steric role
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

substitution (not covered by the Ingold mechanisms), decoordination from a metal ion, coordination to a metal ion, cofactor used, proton transfer, electron transfer, decarboxylation, colligation, intermediate formation, overall product formed

References

  1. Burrell MR et al. (2007), Biochemistry, 46, 12327-12336. Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282,000 by mutating an active site lid. DOI:10.1021/bi700947s. PMID:17924657.
  2. Zhu W et al. (2016), Biochemistry, 55, 2163-2173. Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase. DOI:10.1021/acs.biochem.6b00043. PMID:27014926.

Step 1. Monodentate coordination of oxalate monoanion, displacing one solvent molecule from the Mn(II) centre.

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Catalytic Residues Roles

Residue Roles
Val77A steric role
Met149A steric role
Glu95A metal ligand
His137A metal ligand
His88A metal ligand
His90A metal ligand
Gln139A hydrogen bond acceptor
Asn75A hydrogen bond acceptor
Gln139A hydrogen bond donor
Asn85A hydrogen bond donor

Chemical Components

substitution (not covered by the Ingold mechanisms), decoordination from a metal ion, coordination to a metal ion, cofactor used

Step 2. Dioxygen binding to Mn(II) displaces another solvent molecule, and forms an Mn(III) superoxide metalloradical complex.

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Catalytic Residues Roles

Residue Roles
Gln139A hydrogen bond acceptor
Asn75A hydrogen bond acceptor
Gln139A hydrogen bond donor
Asn85A hydrogen bond donor
Glu95A metal ligand
His137A metal ligand
His88A metal ligand
His90A metal ligand

Chemical Components

cofactor used, decoordination from a metal ion, coordination to a metal ion, substitution (not covered by the Ingold mechanisms)

Step 3. Activation of the Mn(III)-bound oxalate through deprotonation (inferred by curator). This causes the oxalate bridging oxygen to transfer an electron to the Mn(III) centre.

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Catalytic Residues Roles

Residue Roles
Gln139A hydrogen bond donor
Asn85A hydrogen bond donor
Gln139A hydrogen bond acceptor
Asn75A hydrogen bond acceptor
Glu95A metal ligand
His137A metal ligand
His88A metal ligand
His90A metal ligand

Chemical Components

cofactor used, proton transfer, electron transfer

Step 4. Decarboxylation of the radical oxalyl anion.

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Catalytic Residues Roles

Residue Roles

Chemical Components

decarboxylation

Step 5. The superoxide radical binds to the the radical carbonyl group.

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Catalytic Residues Roles

Residue Roles

Chemical Components

colligation, intermediate formation

Step 6. A second decarboxylation occurs and along with two proton transfers, to form hydrogen peroxide.

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Catalytic Residues Roles

Residue Roles

Chemical Components

decarboxylation, proton transfer, overall product formed
Download: Image, Marvin File

Step 1. Monodentate coordination of oxalate monoanion, displacing one solvent molecule from the Mn(II) centre.

Step 2. Dioxygen binding to Mn(II) displaces another solvent molecule, and forms an Mn(III) superoxide metalloradical complex.

Step 3. Activation of the Mn(III)-bound oxalate through deprotonation (inferred by curator). This causes the oxalate bridging oxygen to transfer an electron to the Mn(III) centre.

Step 4. Decarboxylation of the radical oxalyl anion.

Step 5. The superoxide radical binds to the the radical carbonyl group.

Step 6. A second decarboxylation occurs and along with two proton transfers, to form hydrogen peroxide.

Products.

Contributors

Noa Marson, Antonio Ribeiro