Enzyme - Xanthine dehydrogenase

Alternative Name(s)
  • Xanthine oxidoreductase.
  • Xanthine/NAD(+) oxidoreductase.
  • NAD-xanthine dehydrogenase.
  • Xanthine-NAD oxidoreductase.

Catalytic Activity

H2O + NAD(+) + xanthine = H(+) + NADH + urate


FAD; Iron-sulfur; Mo cation.

Reaction Mechanisms

    Mammalian xanthine oxidoreductase catalyses the hydroxylation of hypoxanthine and xanthine, the last two stages in the biosynthesis pathway of urate. The enzyme is first formed as xanthine dehydrogenase (XDH), and exists mostly in this form within the cell but it can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis.

    The active form of the enzyme is a homodimer of a three chain subunits, where both units contain one molybdenum centre, one flavin (FAD) centre and two Fe-S clusters. The oxidation of xanthine to uric acid takes place at the molybdenum centre and results in a two electron reduction of the metal from Mo(VI) to Mo(IV). The enzyme is subsequently reoxidised by molecular oxygen in a reaction that occurs at the FAD cofactor.

    Xanthine dehydrogenase (EC and xanthine oxidase (EC - M-CSA ID:987) are two variants of the same gene product. The former prefers NAD+ as the oxidising substrate whereas the latter uses dioxygen exclusively.

    The most recently proposed mechanism describes a catalytically labile Mo-OH group of the oxidised Mo(VI) enzyme initiating catalysis by base assisted nucleophilic attack on the carbon of the centre to be hydroxylated, with concomitant hydride transfer. This yields a reduced Mo(IV)-SH, derived from the Mo(VI)=S of the oxidised enzyme, with the product remaining coordinated to the molybdenum via the newly introduced hydroxyl group. The product is displaced by attack of a water molecule at the Mo centre. Glu-1261 is well positioned to play the role of the general base in abstracting a proton from the Mo-OH group, while Arg880 stabilises the anionic charge on the substrate while Glu802 is implicated in stabilising the desired product tautomer.

    The mechanism shown here strongly supports all the available evidence. The pH dependence of the reaction strongly suggests that the active site base (Glu1261A) is initially in its unprotonated state and that the enzyme only works on the substrate in its neutral state [PMID:15134930]. The mechanism shown in here is supported by mutational spectroscopic computational and crystallographic studies [PMID:15148401, PMID:15581570, PMID:15134930, PMID:15265866]. There has been some debate as to whether the catalytically labile oxygen is from the Mo=O or Mo-OH groups. Isotope labelling experiments of solvent shows that the catalytically labile group is the Mo-OH and that it is replenished from solvent at the end of the catalytic cycle [PMID:15148401, PMID:15581570].

    Catalytic Residues
    AA Uniprot Uniprot Resid PDB PDB Resid
    Glu P80457 802 1v97 802
    Arg P80457 880 1v97 880
    Glu P80457 1261 1v97 1261
    Step Components

    cofactor used, radical termination, overall reactant used, overall product formed, native state of cofactor regenerated, bimolecular nucleophilic substitution, electron transfer, decoordination from a metal ion, hydride transfer, aromatic bimolecular nucleophilic addition, radical formation, native state of enzyme regenerated, intermediate formation, electron relay, coordination to a metal ion, intermediate terminated, bimolecular nucleophilic addition, proton transfer, redox reaction, aromatic unimolecular elimination by the conjugate base

    Step 1.

    Glu1261 deprotonates the MTE bound hydroxide, activating it for nucleophilic attack upon the xanthine substrate, resulting in a nucleophilic addition of the substrate to the cofactor. Xanthine then transfers a hydride to the sulfur bound to the molybdenum ion (Mo=S) resulting in a two electron reduction of the Mo(VI) to Mo(IV) and a thiol bound to molybdenum.

    Step 2.

    Water deprotonates the molybdenum bound thiol, reforming the Mo=S species and a single electron transfer from the Mo(IV) through the rest of the cofactor and two iron-sulfur clusters to a bound FAD cofactor, forming Mo(V) and a radical on the FAD cofactor.

    Step 3.

    A free hydroxide ion attacks the Mo(V), releasing the urate product (oxidised xanthine), which is re-protonated from the Glu1261, in a nucleophilic substitution reaction. This also results in the second electron transfer from the Mo(V) through the rest of the cofactor and two iron-sulfur clusters to a bound FAD cofactor, which then deprotonates a hydroxonium ion.

    Step 4.

    FAD is regenerated through a hydroxide transfer from the FAD cofactor to the substrate NAD


    The products of the reaction.

Reaction Parameters

  • Kinetic Parameters
    Organism KM Value [mM] Substrate Comment
    Escherichia coli 200 benzaldehyde mutant R440K, pH 4.0, 22°C
    Rhodobacter capsulatus 230 xanthine pH 8.5, 40°C, Split166 mutant
    Acinetobacter baumannii 2740 xanthine pH and temperature not specified in the publication
  • Temperature
    Organism Temperature Range Comment
    Gallus gallus 25 - 41
  • pH
    Organism pH Range Comment
    Homo sapiens 6 - 9.2 trends to increasing activities at higher values
    Arabidopsis thaliana 6.6 - 8
    Gallus gallus 7.2 - 8.7

Associated Proteins

Protein name Organism
Xanthine dehydrogenase FAD-binding subunit Escherichia coli O157:H7
Xanthine dehydrogenase Fruit fly
Xanthine dehydrogenase molybdenum-binding subunit Escherichia coli O157:H7
Probable xanthine dehydrogenase subunit A Bacillus subtilis (strain 168)
Xanthine dehydrogenase 2 Mouse-ear cress