Aldehyde oxidase

 

Aldehyde oxidase is a member of the xanthine oxidase family. It is a complex molybdoenzyme containing a Mo cofactor (Moco), flavin adenine nucleotide (FAD) and two Fe2S2 clusters. The enzyme has broad substrate specificity and catalyses a wide range of reactions. This includes the oxidation of aldehydes and several N-heterocycles, as well as the reduction of some substrates. In the human liver, the enzyme plays an essential role in the detoxification of xenobiotics and contributes to the hepatic clearance of many other drugs.

 

Reference Protein and Structure

Sequence
Q06278 UniProt (1.2.3.1, 1.17.3.-) IPR014313 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
4uhx - Human aldehyde oxidase in complex with phthalazine and thioridazine (2.7 Å) PDBe PDBsum 4uhx
Catalytic CATH Domains
(see all for 4uhx)
Cofactors
Di-mu-sulfido-diiron(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:1.2.3.1)

sulfurated eukaryotic molybdenum cofactor(2-)
CHEBI:60102ChEBI
+
phthalazine
CHEBI:36597ChEBI
+
FAD
CHEBI:16238ChEBI
+
hydron
CHEBI:15378ChEBI
→
phthalazin-1(2H)-one
CHEBI:34023ChEBI
+
CHEBI:X00679X00679
+
FADH2
CHEBI:17877ChEBI
Alternative enzyme names: Quinoline oxidase, Retinal oxidase,

Enzyme Mechanism

Introduction

The step-wise mechanism of oxidation of phthalazine (PHT) to phthalazin-1(2H)-one by hAOX1 begins with protonation of the substrate's N2 atom by Lys893. The hydroxyl group of molybdenum cofactor (Moco) subsequently attacks the substrate, and then a hydride transfer from the substrate to the sulphur atom of the Moco occurs. The rate-limiting step is the hydride transfer, determined by calculated free energy profiles.

Catalytic Residues Roles

UniProt PDB* (4uhx)
Glu1270 Glu1270A Deprotonates Moco, facilitating its nucleophilic attack on substrate. electrostatic interaction, proton acceptor
Lys893 Lys893A Protonates substrate, activating it towards nucleophilic attack by Moco. electrostatic interaction, 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, intermediate formation, bimolecular nucleophilic addition, hydride transfer, rate-determining step, electron transfer, intermediate collapse, cofactor used, redox reaction, inferred reaction step, radical formation, overall product formed, radical termination

References

  1. Ferreira P et al. (2020), ACS Catal, 10, 9276-9286. Catalytic Mechanism of Human Aldehyde Oxidase. DOI:10.1021/acscatal.0c02627.
  2. Mirzaei S et al. (2016), RSC Adv, 6, 109672-109680. Mechanistic study of allopurinol oxidation using aldehyde oxidase, xanthine oxidase and cytochrome P450 enzymes. DOI:10.1039/C6RA19197E.
  3. Coelho C et al. (2015),Human aldehyde oxidase in complex with phthalazine and thioridazine. DOI:10.2210/pdb4uhx/pdb.
  4. Cerqueira NM et al. (2015), J Biol Inorg Chem, 20, 209-217. Insights into the structural determinants of substrate specificity and activity in mouse aldehyde oxidases. DOI:10.1007/s00775-014-1198-2. PMID:25287365.
  5. Yamaguchi Y et al. (2007), J Biochem, 141, 513-524. Human xanthine oxidase changes its substrate specificity to aldehyde oxidase type upon mutation of amino acid residues in the active site: roles of active site residues in binding and activation of purine substrate. DOI:10.1093/jb/mvm053. PMID:17301077.

Step 1. Proton transfer from Lys893 to PHT. The transfer is barrierless and activates PHT for subsequent nucleophilic attack by the hydroxyl oxygen of the Moco (VI).

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Catalytic Residues Roles

Residue Roles
Lys893A proton donor
Glu1270A electrostatic interaction

Chemical Components

proton transfer

Step 2. Deprotonation of the hydroxyl group of the Moco (VI) by Glu1270 and nucleophilic attack on substrate, forming a tetrahedral intermediate.

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Catalytic Residues Roles

Residue Roles
Lys893A electrostatic interaction
Glu1270A proton acceptor

Chemical Components

proton transfer, intermediate formation, ingold: bimolecular nucleophilic addition

Step 3. Hydride transfer from substrate to the Moco (VI). This is the rate-limiting step of the reaction and reduces the Mo to (IV) .

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Catalytic Residues Roles

Residue Roles

Chemical Components

hydride transfer, rate-determining step, electron transfer, intermediate collapse

Step 4. Single electron reduction and protonation of FAD by Moco (IV) forming Moco (V). Mechanism inferred by curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

cofactor used, redox reaction, electron transfer, proton transfer, inferred reaction step, radical formation

Step 5. Second single electron transfer and proton transfer from water forming FADH2 and oxidising Moco (V) to Moco (VI). Mechanism inferred by curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

proton transfer, overall product formed, cofactor used, inferred reaction step, electron transfer, radical termination, redox reaction
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Step 1. Proton transfer from Lys893 to PHT. The transfer is barrierless and activates PHT for subsequent nucleophilic attack by the hydroxyl oxygen of the Moco (VI).

Step 2. Deprotonation of the hydroxyl group of the Moco (VI) by Glu1270 and nucleophilic attack on substrate, forming a tetrahedral intermediate.

Step 3. Hydride transfer from substrate to the Moco (VI). This is the rate-limiting step of the reaction and reduces the Mo to (IV) .

Step 4. Single electron reduction and protonation of FAD by Moco (IV) forming Moco (V). Mechanism inferred by curator.

Step 5. Second single electron transfer and proton transfer from water forming FADH2 and oxidising Moco (V) to Moco (VI). Mechanism inferred by curator.

Products.

Introduction

Concerted mechanism. Moco-bound OH makes a nucleophilic attack on the carbon atom of the substrate, whilst the H atom is transferred to the S atom of the Moco. Supported by kinetic isotopic effects.

Catalytic Residues Roles

UniProt PDB* (4uhx)
Glu1270 Glu1270A Enhances stability of transition state and lowers activation energy of reaction. electrostatic stabiliser
Lys893 Lys893A 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, hydride transfer, radical formation, inferred reaction step, proton transfer, electron transfer, redox reaction, cofactor used, radical termination, overall product formed, keto-enol tautomerisation

References

  1. Montefiori M et al. (2017), ACS Omega, 2, 4237-4244. Aldehyde Oxidase: Reaction Mechanism and Prediction of Site of Metabolism. DOI:10.1021/acsomega.7b00658. PMID:30023718.
  2. Ferreira P et al. (2020), ACS Catal, 10, 9276-9286. Catalytic Mechanism of Human Aldehyde Oxidase. DOI:10.1021/acscatal.0c02627.
  3. Alfaro JF et al. (2008), J Org Chem, 73, 9469-9472. Studies on the mechanism of aldehyde oxidase and xanthine oxidase. DOI:10.1021/jo801053u. PMID:18998731.

Step 1. Moco-bound OH makes a nucleophilic attack on the carbon atom of the substrate, whilst hydride transfer occurs to the S atom of the Moco. Reduces Moco (VI) to Moco (IV).

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Catalytic Residues Roles

Residue Roles
Lys893A electrostatic stabiliser
Glu1270A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, hydride transfer

Step 2. Single electron reduction and protonation of FAD by Moco (IV) forming Moco (V). Mechanism inferred by curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

radical formation, inferred reaction step, proton transfer, electron transfer, redox reaction, cofactor used

Step 3. Second single electron transfer and proton transfer from water forming FADH2 and oxidising Moco (V) to Moco (VI). Mechanism inferred by curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

redox reaction, radical termination, electron transfer, inferred reaction step, cofactor used, overall product formed, proton transfer

Step 4. Keto-enol tautomerisation. Inferred by curator.

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Catalytic Residues Roles

Residue Roles

Chemical Components

keto-enol tautomerisation, inferred reaction step
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Step 1. Moco-bound OH makes a nucleophilic attack on the carbon atom of the substrate, whilst hydride transfer occurs to the S atom of the Moco. Reduces Moco (VI) to Moco (IV).

Step 2. Single electron reduction and protonation of FAD by Moco (IV) forming Moco (V). Mechanism inferred by curator.

Step 3. Second single electron transfer and proton transfer from water forming FADH2 and oxidising Moco (V) to Moco (VI). Mechanism inferred by curator.

Step 4. Keto-enol tautomerisation. Inferred by curator.

Products.

Contributors

Yordanos Abeje, Noa Marson