Xanthine dehydrogenase (mammalian)

 

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 1.17.1.4) and xanthine oxidase (EC 1.17.3.2 - 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.

 

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
1v97 - Crystal Structure of Bovine Milk Xanthine Dehydrogenase FYX-051 bound form (1.94 Å) PDBe PDBsum 1v97
Catalytic CATH Domains
3.30.365.10 CATHdb (see all for 1v97)
Cofactors
Di-mu-sulfido-diiron(2+) (2), Molybdopterin (1), Dioxothiomolybdenum(vi) ion (1), Fadh2(2-) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.17.1.4)

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

Enzyme Mechanism

Introduction

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 Roles

UniProt PDB* (1v97)
Glu802 Glu802A The residue is thought to influence the tautomerisation step, relaying a proton from Glu1261 to a nitrogen on the substrate by lowering the energy of the desired tautomer, with concomitant hydride transfer to the metal cofactor. hydrogen bond acceptor, electrostatic stabiliser
Arg880 Arg880A The positively charged gaunidinium side chain acts to stabilise the build up of negative charge on the substrate once nucleophilic attack has occurred. hydrogen bond donor, electrostatic stabiliser
Glu1261 Glu1261A hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, 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

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

References

  1. Choi EY et al. (2004), J Inorg Biochem, 98, 841-848. Studies on the mechanism of action of xanthine oxidase. DOI:10.1016/j.jinorgbio.2003.11.010. PMID:15134930.
  2. Reschke S et al. (2017), Inorg Chem, 56, 2165-2176. Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X-ray Absorption Spectroscopy. DOI:10.1021/acs.inorgchem.6b02846. PMID:28170236.
  3. Cao H et al. (2014), Biochemistry, 53, 533-541. Substrate orientation and specificity in xanthine oxidase: crystal structures of the enzyme in complex with indole-3-acetaldehyde and guanine. DOI:10.1021/bi401465u. PMID:24397336.
  4. Metz S et al. (2010), J Phys Chem B, 114, 1506-1517. QM/MM studies of xanthine oxidase: variations of cofactor, substrate, and active-site Glu802. DOI:10.1021/jp909999s. PMID:20050623.
  5. Pauff JM et al. (2009), J Biol Chem, 284, 8760-8767. Substrate Orientation and Catalysis at the Molybdenum Site in Xanthine Oxidase: CRYSTAL STRUCTURES IN COMPLEX WITH XANTHINE AND LUMAZINE. DOI:10.1074/jbc.m804517200. PMID:19109252.
  6. Metz S et al. (2009), J Am Chem Soc, 131, 14885-14902. A combined QM/MM study on the reductive half-reaction of xanthine oxidase: substrate orientation and mechanism. DOI:10.1021/ja9045394. PMID:19788181.
  7. Hille R (2005), Arch Biochem Biophys, 433, 107-116. Molybdenum-containing hydroxylases. DOI:10.1016/j.abb.2004.08.012. PMID:15581570.
  8. Zhang XH et al. (2005), Inorg Chem, 44, 1466-1471. A Theoretical Study on the Mechanism of the Reductive Half-Reaction of Xanthine Oxidase. DOI:10.1021/ic048730l. PMID:15732988.
  9. Okamoto K et al. (2004), Proc Natl Acad Sci U S A, 101, 7931-7936. The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition. DOI:10.1073/pnas.0400973101. PMID:15148401.
  10. 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.
  11. Stockert AL et al. (2002), J Am Chem Soc, 124, 14554-14555. The Reaction Mechanism of Xanthine Oxidase:  Evidence for Two-Electron Chemistry Rather Than Sequential One-Electron Steps. DOI:10.1021/ja027388d.

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.

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Catalytic Residues Roles

Residue Roles
Glu1261A hydrogen bond acceptor, electrostatic stabiliser
Arg880A hydrogen bond donor, electrostatic stabiliser
Glu802A hydrogen bond acceptor, electrostatic stabiliser
Glu1261A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, hydride transfer, ingold: bimolecular nucleophilic addition, overall reactant used, cofactor used, intermediate formation

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.

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Catalytic Residues Roles

Residue Roles
Glu1261A hydrogen bond donor, electrostatic stabiliser
Arg880A hydrogen bond donor
Glu802A hydrogen bond acceptor

Chemical Components

proton transfer, redox reaction, radical formation, cofactor used, native state of cofactor regenerated, intermediate formation, overall product formed, electron relay

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.

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Catalytic Residues Roles

Residue Roles
Glu1261A hydrogen bond donor
Arg880A hydrogen bond donor, electrostatic stabiliser
Glu802A hydrogen bond acceptor, electrostatic stabiliser
Glu1261A proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, electron transfer, radical termination, overall reactant used, cofactor used, native state of cofactor regenerated, coordination to a metal ion, decoordination from a metal ion, intermediate terminated, intermediate formation, overall product formed, electron relay

Step 4. FAD is regenerated through a hydroxide transfer from the FAD cofactor to the substrate NAD

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Catalytic Residues Roles

Residue Roles
Glu1261A hydrogen bond acceptor

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, hydride transfer, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, intermediate terminated, overall product formed, native state of enzyme regenerated, native state of cofactor regenerated
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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

Products.

Introduction

An alternative mechanism has been proposed for the formation of the first intermediate in which the mechanism of the reaction proceeds via individual one-electron steps (rather than the obligatory two-electron chemistry of a nucleophilic attack mechanism). However, the lack of inverse relationship between the one electron reduction potential for the purine substrates and the rate of catalysis suggests that the mechanism proceeds via the nucleophilic attack shown in the other proposal.

Catalytic Residues Roles

UniProt PDB* (1v97)
Glu802 Glu802A The residue is thought to influence the tautomerisation step, relaying a proton from Glu1261 to a nitrogen on the substrate by lowering the energy of the desired tautomer, with concomitant hydride transfer to the metal cofactor. hydrogen bond acceptor, electrostatic stabiliser
Arg880 Arg880A The positively charged gaunidinium side chain acts to stabilise the build up of negative charge on the substrate once nucleophilic attack has occurred. hydrogen bond donor, electrostatic stabiliser
Glu1261 Glu1261A Thought to stabilise the reactive intermediates and transition states formed during the course of the reaction in this proposal. hydrogen bond acceptor, 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

bimolecular nucleophilic addition, overall reactant used, cofactor used, intermediate formation, proton transfer, intramolecular nucleophilic addition, electron transfer, hydride transfer, overall product formed, inferred reaction step

References

  1. Stockert AL et al. (2002), J Am Chem Soc, 124, 14554-14555. The Reaction Mechanism of Xanthine Oxidase:  Evidence for Two-Electron Chemistry Rather Than Sequential One-Electron Steps. DOI:10.1021/ja027388d.
  2. Howes BD et al. (1996), Biochemistry, 35, 1432-1443. Evidence favoring molybdenum-carbon bond formation in xanthine oxidase action: 17Q- and 13C-ENDOR and kinetic studies. DOI:10.1021/bi9520500. PMID:8634273.

Step 1. The sulfur of MTE abstracts a proton from the substrate, which initiates a nucleophilic attack on the Mo(VI) to form Mo(IV) and a Mo-C bond.

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Catalytic Residues Roles

Residue Roles
Glu1261A hydrogen bond acceptor, electrostatic stabiliser
Arg880A hydrogen bond donor, electrostatic stabiliser
Glu802A hydrogen bond acceptor, electrostatic stabiliser

Chemical Components

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

Step 2. The hydroxide ligand of the Mo cofactor attacks the carbon of the Mo-C bond, forming a three centre, two electon bond between Mo-C-O and a negatively charged nitrogen which is stabilised by the argenine residue.

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Catalytic Residues Roles

Residue Roles
Glu802A electrostatic stabiliser
Arg880A electrostatic stabiliser
Glu1261A electrostatic stabiliser

Chemical Components

ingold: intramolecular nucleophilic addition

Step 3. A single proton and electron are lost from the Mo-thiol group and substrate.

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Catalytic Residues Roles

Residue Roles
Glu802A electrostatic stabiliser
Arg880A electrostatic stabiliser
Glu1261A electrostatic stabiliser

Chemical Components

electron transfer, hydride transfer, overall product formed, overall reactant used, inferred reaction step

Step 4. The negatively charged nitrogen initiates a proton transfer from the active site water to the oxygen atom now bonded to the C8 of the intermediate.

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Catalytic Residues Roles

Residue Roles
Glu802A electrostatic stabiliser
Arg880A electrostatic stabiliser
Glu1261A electrostatic stabiliser

Chemical Components

overall product formed, inferred reaction step, electron transfer, proton transfer
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Step 1. The sulfur of MTE abstracts a proton from the substrate, which initiates a nucleophilic attack on the Mo(VI) to form Mo(IV) and a Mo-C bond.

Step 2. The hydroxide ligand of the Mo cofactor attacks the carbon of the Mo-C bond, forming a three centre, two electon bond between Mo-C-O and a negatively charged nitrogen which is stabilised by the argenine residue.

Step 3. A single proton and electron are lost from the Mo-thiol group and substrate.

Step 4. The negatively charged nitrogen initiates a proton transfer from the active site water to the oxygen atom now bonded to the C8 of the intermediate.

Products.

Introduction

A third proposal suggests that the first intermediate is formed via the addition of the C8-H bond across the Mo=O group of the molybdenum centre. However, the Mo-O-C bond in the product of this step has been crystallographically proven which rules out this alternative.

Catalytic Residues Roles

UniProt PDB* (1v97)
Glu802 Glu802A The residue is thought to influence the tautomerisation step. hydrogen bond acceptor, electrostatic stabiliser
Arg880 Arg880A The positively charged gaunidinium side chain acts to stabilise the build up of negative charge on the substrate once nucleophilic attack has occurred. hydrogen bond donor, electrostatic stabiliser
Glu1261 Glu1261A Helps stabilise the reactive intermediates and transition states formed during the course of the reaction. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser, 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

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

References

  1. Xia M et al. (1999), J Biol Chem, 274, 3323-3330. The Reductive Half-reaction of Xanthine Oxidase. DOI:10.1074/jbc.274.6.3323.

Step 1. The sulfur of the the MTE cofactor abstracts the proton from the C8 of the substrate. This initiates a nucleophilic attack upon the Mo=O ligand, resulting in a two electron reduction of the Mo(VI) to Mo(IV) and a thiol bound to molybdenum.

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Catalytic Residues Roles

Residue Roles
Glu1261A hydrogen bond acceptor, electrostatic stabiliser
Arg880A hydrogen bond donor, electrostatic stabiliser
Glu802A hydrogen bond acceptor, electrostatic stabiliser

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, hydride transfer, ingold: bimolecular nucleophilic addition, overall reactant used, cofactor used, intermediate formation

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.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu1261A hydrogen bond donor, electrostatic stabiliser
Arg880A hydrogen bond donor
Glu802A hydrogen bond acceptor

Chemical Components

proton transfer, redox reaction, radical formation, cofactor used, native state of cofactor regenerated, intermediate formation, overall product formed, electron relay

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.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu1261A hydrogen bond donor
Arg880A hydrogen bond donor, electrostatic stabiliser
Glu802A hydrogen bond acceptor, electrostatic stabiliser
Glu1261A proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, electron transfer, radical termination, overall reactant used, cofactor used, native state of cofactor regenerated, coordination to a metal ion, decoordination from a metal ion, intermediate terminated, intermediate formation, overall product formed, electron relay

Step 4. FAD is regenerated through a hydroxide transfer from the FAD cofactor to the substrate NAD

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Catalytic Residues Roles

Residue Roles
Glu1261A hydrogen bond acceptor

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, hydride transfer, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, intermediate terminated, overall product formed, native state of enzyme regenerated, native state of cofactor regenerated
Download: Image, Marvin File

Step 1. The sulfur of the the MTE cofactor abstracts the proton from the C8 of the substrate. This initiates a nucleophilic attack upon the Mo=O ligand, 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

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, James W. Murray, Craig Porter