Xanthine dehydrogenase (bacterial)

 

Rhodobacter capsulatus xanthine dehydrogenase (RcXDH), a cytoplasmic enzyme, catalyses the hydroxylation of a wide variety of purine, pyrimidine, pterin, and aldehyde substrates. Oxidation of the substrate occurs in the molybdenum centre, and the reduced enzyme is reoxidised by the oxidant substrate, NAD+ or molecular oxygen, through FAD. The cofactors of this enzyme are located on different polypeptides. The FAD and the two different [2Fe-2S] iron-sulphur clusters are found on the XDHA subunit, while the molybdenum cofactor (Moco), with a bicyclic pterin and monocyclic pyran coordinated to it, is bound to the XDHB subunit.

 

Reference Protein and Structure

Sequences
O54050 UniProt (1.1.1.204)
O54051 UniProt (1.1.1.204) IPR014309 (Sequence Homologues) (PDB Homologues)
Biological species
Rhodobacter capsulatus (Bacteria) Uniprot
PDB
1jrp - Crystal Structure of Xanthine Dehydrogenase inhibited by alloxanthine from Rhodobacter capsulatus (3.0 Å) PDBe PDBsum 1jrp
Catalytic CATH Domains
3.30.365.10 CATHdb (see all for 1jrp)
Cofactors
Di-mu-sulfido-diiron(2+) (2), Dioxothiomolybdenum(vi) ion (1), Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.17.1.4)

water
CHEBI:15377ChEBI
+
NAD(1-)
CHEBI:57540ChEBI
+
9H-xanthine
CHEBI:17712ChEBI
→
hydron
CHEBI:15378ChEBI
+
NADH(2-)
CHEBI:57945ChEBI
+
7,9-dihydro-1H-purine-2,6,8(3H)-trione
CHEBI:17775ChEBI
Alternative enzyme names: NAD-xanthine dehydrogenase, Xanthine oxidoreductase, Xanthine-NAD oxidoreductase, Xanthine/NAD(+) oxidoreductase,

Enzyme Mechanism

Introduction

XDH cycles between mainly 2-electron and 4-electron reduced states during enzyme turnover. Catalysis is initiated by the abstraction of a proton from the Mo-OH group by Glu730, followed by a nucleophilic attack on the carbon centre to be hydroxylated (the C-8 position of the substrate) and the concomitant hydride transfer from the C-8 carbon centre to the Mo(VI)=S of the molybdenum centre. This yields an LMo(IV)(SH)(OR) transition state intermediate, with L representing a unique pyranopterin cofactor coordinated to the metal via an enedithiolate sidechain, and OR representing the now hydroxylated product coordinated to the molybdenum via the newly introduced hydroxyl group. The accumulation of negative charge on the transition state is stabilised by Arg310. The bound product is displaced from the molybdenum coordination sphere by hydroxide from the solvent, electron transfer out of the molybdenum centre to FAD via the iron-sulphur centres, and deprotonation of the Mo-SH to regenerate the original, oxidised LMo(VI)OS(OH). The iron-sulphur centres, as well as providing an electron transfer pathway from molybdenum to FAD, also act as electron sinks, storing reducing equivalents during catalysis, and so control the reactivity of FAD by way of its one-electron-reduced or fully-reduced state. Electron transfer to FAD from the iron-sulphur centre occurs by one-electron steps, followed by a two-electron transfer to react with NAD+. After binding of NAD+, fully reduced FAD is oxidised concomitantly with the reduction of NAD+.

Catalytic Residues Roles

UniProt PDB* (1jrp)
Glu730 Glu730B Glu730 acts as an essential base catalyst initiating substrate oxidation by abstracting a proton from the hydroxyl group of the molybdenum centre. proton shuttle (general acid/base)
Arg310 Arg310B Arg310 stabilises the accumulation of negative charge in the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position of the substrate. electrostatic stabiliser
*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

References

  1. Pauff JM et al. (2007), J Biol Chem, 282, 12785-12790. The Role of Arginine 310 in Catalysis and Substrate Specificity in Xanthine Dehydrogenase from Rhodobacter capsulatus. DOI:10.1074/jbc.m700364200. PMID:17327224.
  2. Du Y et al. (2018), J Organomet Chem, 864, 58-67. Computational exploration of reactive fragment for mechanism-based inhibition of xanthine oxidase. DOI:10.1016/j.jorganchem.2018.01.018.
  3. Hall J et al. (2014), J Biol Chem, 289, 32121-32130. The reductive half-reaction of xanthine dehydrogenase from Rhodobacter capsulatus: the role of Glu232 in catalysis. DOI:10.1074/jbc.M114.603456. PMID:25258317.
  4. Leimkühler S et al. (2004), J Biol Chem, 279, 40437-40444. The Role of Active Site Glutamate Residues in Catalysis ofRhodobacter capsulatusXanthine Dehydrogenase. DOI:10.1074/jbc.m405778200. PMID:15265866.
  5. Truglio JJ et al. (2002), Structure, 10, 115-125. Crystal structures of the active and alloxanthine-inhibited forms of xanthine dehydrogenase from Rhodobacter capsulatus. PMID:11796116.
  6. Harris CM et al. (1999), J Biol Chem, 274, 4561-4569. Role of the Flavin Midpoint Potential and NAD Binding in Determining NAD Versus Oxygen Reactivity of Xanthine Oxidoreductase. DOI:10.1074/jbc.274.8.4561. PMID:9988690.
  7. Hunt J et al. (1994), J Biol Chem, 269, 18904-18914. Studies of the reductive half-reaction of milk xanthine dehydrogenase. PMID:8034647.
  8. Olson JS et al. (1974), J Biol Chem, 249, 4363-4382. The mechanism of action of xanthine oxidase. PMID:4367215.

Step 1.

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Catalytic Residues Roles

Residue Roles
Arg310B electrostatic stabiliser
Glu730B proton shuttle (general acid/base)

Chemical Components

Step 1.

Contributors

Gemma L. Holliday