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Catalytic Site Atlas Version 2.2.12
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CSA entry for 1jrp
Original Entry
Title:
Oxidoreductase
Compound:
Xanthine dehydrogenase
Mutant:
No
UniProt/Swiss-Prot:
O54050-O54050
EC Class:
1.1.1.204
Other CSA Entries:
Overview of all sites for 1jrp
Homologues of 1jrp
Entries for UniProt/Swiss-Prot: O54050
Entries for EC: 1.1.1.204
Other Databases:
PDB entry: 1jrp
PDBsum entry: 1jrp
UniProt/Swiss-Prot: O54050
IntEnz entry: 1.1.1.204
Literature Report:
Introduction:
Rhodobacter capsulatus xanthine dehydrogenase (RcXDH) (formerly E.C. 1.1.1.204), 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.

Mechanism:
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+.

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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ARGB 310 0Sidechain
ElectrostaticTransition state
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.
Evidence from paper Evidence concerns Evidence type
PubMed ID 17327224 Current protein Kinetic studies
PubMed ID 17327224 Current protein Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
GLUB 730 0Sidechain
Acid/baseCofactor
Glu730 acts as an essential base catalyst initiating substrate oxidation by abstracting a proton from the hydroxyl group of the molybdenum centre.
Evidence from paper Evidence concerns Evidence type
PubMed ID 15265866 Current protein Kinetic studies
PubMed ID 15265866 Current protein Mutagenesis of residue
PubMed ID 15265866 Current protein Ligand is essential for catalysis

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
I3001 0
Electron donor/acceptorCofactor
One of the two different [2Fe-2S] iron-sulphur clusters, providing an electron transfer pathway from the molybdenum centre to FAD and acting as an electron sink, storing reducing equivalents during catalysis, thus controlling the one-electron reduced / fully reduced state of FAD.
Evidence from paper Evidence concerns Evidence type
PubMed ID 4367215 Related protein: UniProt P80457

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
I3002 0
Electron donor/acceptorCofactor
One of the two different [2Fe-2S] iron-sulphur clusters, providing an electron transfer pathway from the molybdenum centre to FAD and acting as an electron sink storing reducing equivalents during catalysis, thus controlling the one-electron reduced / fully reduced state of FAD
Evidence from paper Evidence concerns Evidence type
PubMed ID 4367215 Related protein: UniProt P80457

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
I3004 0
NucleophileSubstrate
Electron donor/acceptorCofactor
Substrate
The Mo-OH group is deprotonated and attacks the carbon centre on the substrate that is to be hydroxylated. The Mo(VI)=S undergoes hydride transfer from the substrate carbon centre concomitantly with the nucleophilic attack. After the product has been displaced from the intermediate by hydroxide from the solvent, electrons are transferred out of the molybdenum centre to the iron-sulphur centres and Mo-SH is deprotonated.
Evidence from paper Evidence concerns Evidence type
PubMed ID 15265866 Current protein Residue is covalently bound to intermediate, based on structural data
PubMed ID 4367215 Related protein: UniProt P80457

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
I3005 0
Substrate
FAD accepts electrons from the iron-sulphur cluster (3002) to become fully reduced FADH2. NAD+ binds and hydride transfer from FADH2 to NAD+ results in the formation of NADH and FAD.
Evidence from paper Evidence concerns Evidence type
PubMed ID 8034647 Related protein: UniProt P80457
PubMed ID 9988690 Related protein: UniProt P80457 Kinetic studies
Notes:



References:
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