Xanthine oxidase

 

Xanthine oxidase (XO) is a homodimer enzyme that gives its name to the xanthine oxidase structural family. XO catalyses the conversion of xanthine (XAN) to uric acid (URC) using a molybdenum cofactor (Moco). This is the last step of purine nucleotide catabolism in humans as well as primates, birds, reptiles and insects.

Xanthine oxidase comes from the same gene product as xanthine dehydrogenase (XDH) (EC 1.17.1.4 - M-CSA ID:139). XO and XDH share the same basic mechanism but differ in regeneration of the cofactor. Unlike XDH, XO does not use NAD+ as an oxidising substrate and instead uses O2.
The enzyme is of interest due to its relationship with certain diseases. For example, gout and uric acid stone formation are caused by intense activity of xanthine oxidase. Also, xanthinuria is a rare genetic disorder associated with low xanthine oxidase activity.

 

Reference Protein and Structure

Sequence
P80457 UniProt (1.17.1.4, 1.17.3.2) IPR016208 (Sequence Homologues) (PDB Homologues)
Biological species
Bos taurus (Cattle) Uniprot
PDB
3amz - Bovine Xanthine Oxidoreductase urate bound form (2.1 Å) PDBe PDBsum 3amz
Catalytic CATH Domains
3.30.365.10 CATHdb (see all for 3amz)
Cofactors
Dioxothiomolybdenum(vi) ion (1), Molybdopterin (1), Di-mu-sulfido-diiron(2+) (2), Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.17.3.2)

water
CHEBI:15377ChEBI
+
dioxygen
CHEBI:15379ChEBI
+
9H-xanthine
CHEBI:17712ChEBI
→
hydrogen peroxide
CHEBI:16240ChEBI
+
7,9-dihydro-1H-purine-2,6,8(3H)-trione
CHEBI:17775ChEBI
Alternative enzyme names: Schardinger enzyme, Hypoxanthine oxidase, Hypoxanthine-xanthine oxidase, Hypoxanthine:oxygen oxidoreductase, Xanthine oxidoreductase, Xanthine:O(2) oxidoreductase, Xanthine:xanthine oxidase,

Enzyme Mechanism

Introduction

The mechanism begins with proton transfer from the molybdenum cofactor (Moco) to Glu1261 and the activated hydroxyl group makes a nucleophilic attack on the xanthine (XAN) substrate. Then a hydride is transferred from the tetrahedral intermediate to the sulfur atom of the Moco, reducing Mo(VI) to Mo(IV). Protonation by Arg880 forms uric acid (URC) which can then leave the active site. The Moco is oxidised using electron transfer to FAD, which is subsequently regenerated using O2. Coordination of a water molecule to the Moco completes the turnover. There are other mechanistic proposals available in the literature.

Catalytic Residues Roles

UniProt PDB* (3amz)
Glu1261 Glu1261A Glu1261 abstracts a proton from the hydroxyl group that is coordinated to the Mo(VI) ion. This activates the oxygen for a nucleophilic attack on the carbon atom of the xanthine substrate. proton acceptor
Glu802 Glu802A Glu802 has an important role in positioning the substrate and to stabilise the intermediate generated after the first step. electrostatic stabiliser
Arg880 Arg880A Donates a proton to nitrogen of URT, mediated by a water molecule. proton donor
*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

bimolecular nucleophilic addition, cofactor used, proton transfer, intermediate formation, hydride transfer, rate-determining step, overall product formed, radical formation, electron relay, inferred reaction step, radical termination, coordination to a metal ion, native state of cofactor regenerated, electron transfer, overall reactant used, bimolecular homolytic addition, aromatic intramolecular elimination, intermediate terminated, native state of enzyme regenerated

References

  1. Ribeiro PMG et al. (2021), Inorg Chem Front, 8, 405-416. The complete catalytic mechanism of xanthine oxidase: a computational study. DOI:10.1039/d0qi01029d.
  2. Romero E et al. (2018), Chem Rev, 118, 1742-1769. Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes. DOI:10.1021/acs.chemrev.7b00650. PMID:29323892.

Step 1. Glu1261 abstracts a proton from the hydroxyl group that is coordinated to the Mo(VI) ion. This activates the oxygen for a nucleophilic attack on the carbon atom of the xanthine substrate. The negative charge is stabilised via resonance throughout the substrate.

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Catalytic Residues Roles

Residue Roles
Glu802A electrostatic stabiliser
Glu1261A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, cofactor used, proton transfer, intermediate formation

Step 2. Hydride transfer from the carbon atom of the substrate to sulfur on the Moco. Mo(VI) is reduced to Mo(IV).

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Catalytic Residues Roles

Residue Roles
Glu802A electrostatic stabiliser

Chemical Components

hydride transfer, cofactor used, rate-determining step

Step 3. Proton transfer from Arg880 to nitrogen of URT, mediated by a water molecule. At this point, the product can be released from the active site.

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Catalytic Residues Roles

Residue Roles
Arg880A proton donor

Chemical Components

proton transfer, overall product formed

Step 4. The Mo ion is oxidised from Mo(IV) to Mo(V). A single electron is transferred to an FAD cofactor via two [2Fe-2S] clusters. The mechanism inferred by curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

cofactor used, radical formation, electron relay, proton transfer, inferred reaction step

Step 5. The Mo ion is oxidised from Mo(V) to Mo(VI). A single electron is transferred to the FADH radical via two [2Fe-2S] clusters, forming reduced FAD. The mechanism is inferred by the curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

electron relay, cofactor used, radical termination, proton transfer, inferred reaction step

Step 6. A water molecule coordinates to Mo.

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Catalytic Residues Roles

Residue Roles

Chemical Components

coordination to a metal ion

Step 7. The coordinated water molecule is deprotonated, reforming the Moco.

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Catalytic Residues Roles

Residue Roles

Chemical Components

native state of cofactor regenerated, proton transfer

Step 8. Regeneration of FAD begins with double bond rearrangement, causing a single electron to be transferred to a dioxygen molecule.

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Catalytic Residues Roles

Residue Roles

Chemical Components

radical formation, electron transfer, overall reactant used, intermediate formation, inferred reaction step

Step 9. The dioxygen molecule undergoes a homolytic reaction in which it colligates to FAD. Proton transfer from solvent.

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Catalytic Residues Roles

Residue Roles

Chemical Components

radical termination, ingold: bimolecular homolytic addition, proton transfer, intermediate formation, inferred reaction step

Step 10. The peroxo group deprotonates FAD, which initiates the elimination of hydrogen peroxide.

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Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: aromatic intramolecular elimination, inferred reaction step, native state of cofactor regenerated, intermediate terminated, overall product formed, native state of enzyme regenerated
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Step 1. Glu1261 abstracts a proton from the hydroxyl group that is coordinated to the Mo(VI) ion. This activates the oxygen for a nucleophilic attack on the carbon atom of the xanthine substrate. The negative charge is stabilised via resonance throughout the substrate.

Step 2. Hydride transfer from the carbon atom of the substrate to sulfur on the Moco. Mo(VI) is reduced to Mo(IV).

Step 3. Proton transfer from Arg880 to nitrogen of URT, mediated by a water molecule. At this point, the product can be released from the active site.

Step 4. The Mo ion is oxidised from Mo(IV) to Mo(V). A single electron is transferred to an FAD cofactor via two [2Fe-2S] clusters. The mechanism inferred by curator.

Step 5. The Mo ion is oxidised from Mo(V) to Mo(VI). A single electron is transferred to the FADH radical via two [2Fe-2S] clusters, forming reduced FAD. The mechanism is inferred by the curator.

Step 6. A water molecule coordinates to Mo.

Step 7. The coordinated water molecule is deprotonated, reforming the Moco.

Step 8. Regeneration of FAD begins with double bond rearrangement, causing a single electron to be transferred to a dioxygen molecule.

Step 9. The dioxygen molecule undergoes a homolytic reaction in which it colligates to FAD. Proton transfer from solvent.

Step 10. The peroxo group deprotonates FAD, which initiates the elimination of hydrogen peroxide.

Products.

Contributors

Noa Marson, Antonio Ribeiro