Urate oxidase

 

Urate oxidase is an essential enzyme involved in purine degradation pathway. It is responsible for the conversion of uric acid to allantoin, thus catalysing the oxidative ring opening of the purine ring in the degradative pathway.

The enzyme is lacking in humans, it can therefore be used as a protein drug to treat / reduce toxic uric acid accumulation.

The active enzyme is a homotetramer, each dimer can be considered as a porin-like structure, with the 16 antiparallel strands superimposed in a reverse handed manner on those from a porin, the helices taking the place of the membrane. The enzyme was originally thought to require a copper cofactor, however it has since been shown that is doesn't require a cofactor [PMID:12680763].

The enzyme is a member of a family of proteins with multimeric barrels face to face related by two-fold symmetry, proposed to be a new family of tunnel-shaped proteins.

 

Reference Protein and Structure

Sequence
Q00511 UniProt (1.7.3.3) IPR002042 (Sequence Homologues) (PDB Homologues)
Biological species
Aspergillus flavus (Fungus) Uniprot
PDB
1wrr - Urate oxidase from aspergillus flavus complexed with 5-amino 6-nitro uracil (1.64 Å) PDBe PDBsum 1wrr
Catalytic CATH Domains
3.10.270.10 CATHdb (see all for 1wrr)
Click To Show Structure

Enzyme Reaction (EC:1.7.3.3)

dioxygen
CHEBI:15379ChEBI
+
water
CHEBI:15377ChEBI
+
7,9-dihydro-1H-purine-2,6,8(3H)-trione
CHEBI:17775ChEBI
→
hydrogen peroxide
CHEBI:16240ChEBI
+
5-hydroxyisouric acid
CHEBI:18072ChEBI
Alternative enzyme names: Uric acid oxidase, Uricase, Uricase II, Urate oxidase,

Enzyme Mechanism

Introduction

Urate oxidase catalyses the first step of the oxidation of uric acid. This reaction is followed by several uncatalysed steps to give allantoin. There is no prosthetic group or metal ion present in the catalytic reaction. Most of the enzyme's catalytic activity results from isolating its substrates in the optimum geometry for the reaction to occur. Uric acid is stabilised as a dianion by urate oxidase through hydrogen bonds with ARG 176 and GLN 228. GLN 228 is positioned to anchor the purine ring.

Catalytic Residues Roles

UniProt PDB* (1wrr)
Lys11, Thr58, His257 Lys10A, Thr57A, His256A(AA) These residues form a Thr-Lys-His catalytic triad that is responsible for the transfer of protons to and from the active site. All three residues act as general acid/bases. proton relay, hydrogen bond donor, proton acceptor, proton donor
Arg177, Gln229 Arg176A(AA), Gln228A(AA) Help bind and stabilise the substrate in the active site. 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, assisted tautomerisation (not keto-enol), overall reactant used, intermediate formation, proton relay, radical formation, electron transfer, colligation, radical termination, intramolecular elimination, intermediate collapse, overall product formed, rate-determining step, bimolecular electrophilic addition, intermediate terminated, native state of enzyme regenerated

References

  1. Imhoff RD et al. (2003), Biochemistry, 42, 4094-4100. General Base Catalysis in the Urate Oxidase Reaction:  Evidence for a Novel Thr−Lys Catalytic Diad†. DOI:10.1021/bi027377x. PMID:12680763.
  2. Colloc'h N et al. (2014), FEBS Lett, 588, 1715-1719. Functional relevance of the internal hydrophobic cavity of urate oxidase. DOI:10.1016/j.febslet.2014.03.017. PMID:24657440.
  3. Bui S et al. (2014), Angew Chem Int Ed Engl, 53, 13710-13714. Direct Evidence for a Peroxide Intermediate and a Reactive Enzyme-Substrate-Dioxygen Configuration in a Cofactor-free Oxidase. DOI:10.1002/anie.201405485. PMID:25314114.
  4. Oksanen E et al. (2014), PLoS One, 9, e86651-. The Neutron Structure of Urate Oxidase Resolves a Long-Standing Mechanistic Conundrum and Reveals Unexpected Changes in Protonation. DOI:10.1371/journal.pone.0086651. PMID:24466188.
  5. Gabison L et al. (2011), Proteins, 79, 1964-1976. X-ray, ESR, and quantum mechanics studies unravel a spin well in the cofactor-less urate oxidase. DOI:10.1002/prot.23022. PMID:21491497.
  6. Altarsha M et al. (2009), Bioorg Chem, 37, 111-125. Intrinsic reactivity of uric acid with dioxygen: Towards the elucidation of the catalytic mechanism of urate oxidase. DOI:10.1016/j.bioorg.2009.05.004. PMID:19539344.
  7. Colloc'h N et al. (2008), Biophys J, 95, 2415-2422. Oxygen Pressurized X-Ray Crystallography: Probing the Dioxygen Binding Site in Cofactorless Urate Oxidase and Implications for Its Catalytic Mechanism. DOI:10.1529/biophysj.107.122184. PMID:18375516.
  8. Gabison L et al. (2008), BMC Struct Biol, 8, 32-. Structural analysis of urate oxidase in complex with its natural substrate inhibited by cyanide: Mechanistic implications. DOI:10.1186/1472-6807-8-32. PMID:18638417.
  9. Gabison L et al. (2006), FEBS Lett, 580, 2087-2091. Recapture of [S]-allantoin, the product of the two-step degradation of uric acid, by urate oxidase. DOI:10.1016/j.febslet.2006.03.007. PMID:16545381.
  10. Retailleau P et al. (2004), Acta Crystallogr D Biol Crystallogr, 60, 453-462. Complexed and ligand-free high-resolution structures of urate oxidase (Uox) fromAspergillus flavus: a reassignment of the active-site binding mode. DOI:10.1107/s0907444903029718. PMID:14993669.
  11. Colloc'h N et al. (1997), Nat Struct Biol, 4, 947-952. Crystal Structure of the protein drug urate oxidase-inhibitor complex at 2.05 Å resolution. DOI:10.1038/nsb1197-947. PMID:9360612.

Step 1. Water deprotonates His256AA, which deprotonates the urate substrate through Lys10 and Thr57.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr57A hydrogen bond acceptor, proton relay
Lys10A hydrogen bond donor, proton relay
His256A(AA) hydrogen bond acceptor, hydrogen bond donor, proton relay
Gln228A(AA) hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Arg176A(AA) electrostatic stabiliser
Lys10A proton donor, proton acceptor
Thr57A proton acceptor
His256A(AA) proton donor
Thr57A proton donor
His256A(AA) proton acceptor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), overall reactant used, intermediate formation, proton relay

Step 2. The C2 oxyanion collapses initiating a single electron transfer to dioxygen, which deprotonates water.

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Catalytic Residues Roles

Residue Roles
Thr57A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Lys10A hydrogen bond donor
Gln228A(AA) hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Arg176A(AA) electrostatic stabiliser

Chemical Components

radical formation, electron transfer, proton transfer, overall reactant used, intermediate formation

Step 3. The dioxygen radical undergoes a homolytic reaction and colligates to the urate radical.

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Catalytic Residues Roles

Residue Roles
Thr57A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Lys10A hydrogen bond donor
Gln228A(AA) hydrogen bond acceptor, hydrogen bond donor
Arg176A(AA) electrostatic stabiliser

Chemical Components

colligation, radical termination, intermediate formation

Step 4. The peroxo group deprotonates urate hydroperoxide, which initiates the elimination of hydrogen peroxide.

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Catalytic Residues Roles

Residue Roles
Thr57A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Lys10A hydrogen bond donor
Gln228A(AA) hydrogen bond acceptor, hydrogen bond donor
Arg176A(AA) electrostatic stabiliser

Chemical Components

ingold: intramolecular elimination, intermediate collapse, intermediate formation, overall product formed, rate-determining step

Step 5. N7 of the substrate deprotonates water, which initiates a nucleophilic attack on the C5 of the substrate in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Thr57A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Lys10A hydrogen bond donor
Gln228A(AA) hydrogen bond acceptor, hydrogen bond donor
Arg176A(AA) electrostatic stabiliser

Chemical Components

ingold: bimolecular electrophilic addition, overall reactant used, intermediate terminated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Water deprotonates His256AA, which deprotonates the urate substrate through Lys10 and Thr57.

Step 2. The C2 oxyanion collapses initiating a single electron transfer to dioxygen, which deprotonates water.

Step 3. The dioxygen radical undergoes a homolytic reaction and colligates to the urate radical.

Step 4. The peroxo group deprotonates urate hydroperoxide, which initiates the elimination of hydrogen peroxide.

Step 5. N7 of the substrate deprotonates water, which initiates a nucleophilic attack on the C5 of the substrate in an addition reaction.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Christian Drew, Craig Porter