Vanillyl-alcohol oxidase

 

Vanillyl Alcohol Oxidase (VAO) is a broad specificity enzyme belonging to a family of FAD-dependent oxidoreductases. VAO can hydrolyse a variety of substrates but is induced in Penicillium simplicissimum in the presence of 4-(methoxymethol)phenol in the medium, suggesting that this is the main physiological substrate.

 

Reference Protein and Structure

Sequence
P56216 UniProt (1.1.3.38) IPR016170 (Sequence Homologues) (PDB Homologues)
Biological species
Penicillium simplicissimum (Fungus) Uniprot
PDB
1vao - STRUCTURE OF THE OCTAMERIC FLAVOENZYME VANILLYL-ALCOHOL OXIDASE (2.5 Å) PDBe PDBsum 1vao
Catalytic CATH Domains
3.30.465.10 CATHdb 3.30.43.10 CATHdb 3.40.462.10 CATHdb (see all for 1vao)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.1.3.38)

dioxygen
CHEBI:15379ChEBI
+
water
CHEBI:15377ChEBI
+
4-(methoxymethyl)phenol
CHEBI:133885ChEBI
→
hydrogen peroxide
CHEBI:16240ChEBI
+
methanol
CHEBI:17790ChEBI
+
4-hydroxybenzaldehyde
CHEBI:17597ChEBI
Alternative enzyme names: 4-hydroxy-2-methoxybenzyl alcohol oxidase,

Enzyme Mechanism

Introduction

His422 binds the flavin cofactor covalently. Site-directed mutagenesis has shown that Asp170 is essential for catalysis. The substrate is shielded from solvent suggesting a direct hydride transfer mechanism. The substrate is oxidised from the C-alpha atom to the N5 flavin atom. The reduced cofactor is then reoxidised by molecular oxygen, producing hydrogen peroxide. The p-quinone-methoxymethide intermediate is hydroxylated by a water molecule, possibly activated by Asp170. Tyr108, Tyr503 and Arg504 form an anion-binding subsite which stabilises the phenolate form of the substrate.

Catalytic Residues Roles

UniProt PDB* (1vao)
His422 His422A The covalent attachment of FAD to His244 increases the redox potential of the FAD, thus enhancing the rate of the redox reaction [PMID:10585424]. increase redox potential, covalently attached, activator
Arg504 Arg504A Arg504 acts to stabilise the negatively charged flavin. However, upon flavin reoxidation, Arg504 is deprived of its anionic partner, triggering the development of a negative charge on the quinone oxygen atom. Thus increasing the electrophilicity of the methide carbon [PMID:9261083]. activator, hydrogen bond donor, electrostatic stabiliser
Tyr503, Tyr108 Tyr503A, Tyr108A Activate and stablise the reactive intermediate formed during the course of the reaction. hydrogen bond donor, electrostatic stabiliser
Asp170 Asp170A Acts as a general acid/base throughout the reaction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
*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, overall reactant used, intermediate formation, aromatic unimolecular elimination by the conjugate base, hydride transfer, aromatic bimolecular nucleophilic addition, cofactor used, rate-determining step, electron transfer, radical formation, colligation, enzyme-substrate complex formation, aromatic bimolecular elimination, enzyme-substrate complex cleavage, intermediate collapse, native state of cofactor regenerated, overall product formed, bimolecular nucleophilic substitution, hydrolysis, intermediate terminated, native state of enzyme regenerated

References

  1. Mattevi A et al. (1997), Structure, 5, 907-920. Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity. DOI:10.1016/s0969-2126(97)00245-1. PMID:9261083.
  2. Jin J et al. (2008), FEBS J, 275, 5191-5200. Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process. DOI:10.1111/j.1742-4658.2008.06649.x. PMID:18793324.
  3. van den Heuvel RH et al. (2000), J Biol Chem, 275, 14799-14808. Asp-170 Is Crucial for the Redox Properties of Vanillyl-alcohol Oxidase. DOI:10.1074/jbc.275.20.14799. PMID:10809721.
  4. van Den Heuvel RH et al. (2000), Proc Natl Acad Sci U S A, 97, 9455-9460. Inversion of stereospecificity of vanillyl-alcohol oxidase. DOI:10.1073/pnas.160175897. PMID:10920192.
  5. Fraaije MW et al. (1999), J Biol Chem, 274, 35514-35520. Covalent Flavinylation Is Essential for Efficient Redox Catalysis in Vanillyl-alcohol Oxidase. DOI:10.1074/jbc.274.50.35514. PMID:10585424.

Step 1. Asp170 deprotonates the 4-OH of the substrate, forming a pheonlate.

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Catalytic Residues Roles

Residue Roles
His422A increase redox potential
Arg504A hydrogen bond donor
Tyr503A hydrogen bond donor
Tyr108A hydrogen bond donor
Asp170A hydrogen bond acceptor
His422A covalently attached
Asp170A proton acceptor

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 2. The oxyanion collapses with double bond rearrangement, eliminating a hydride from the 1-methoxy carbon, which is added to FAD.

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Catalytic Residues Roles

Residue Roles
Arg504A hydrogen bond donor, electrostatic stabiliser
Tyr503A hydrogen bond donor, electrostatic stabiliser
Tyr108A hydrogen bond donor, electrostatic stabiliser
His422A covalently attached, activator, increase redox potential

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, hydride transfer, ingold: aromatic bimolecular nucleophilic addition, cofactor used, intermediate formation, rate-determining step

Step 3. The FAD undergoes double bond rearrangement which causes a single electron to be transferred to a dioxygen molecule.

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Catalytic Residues Roles

Residue Roles
Arg504A hydrogen bond donor, electrostatic stabiliser
Tyr503A hydrogen bond donor
Tyr108A hydrogen bond donor
Asp170A hydrogen bond donor
His422A covalently attached, activator, increase redox potential

Chemical Components

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

Step 4. The dioxygen molecule undergoes a homolytic reaction in which it colligates to FAD, with concomitant deprotonation of Asp170.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg504A hydrogen bond donor
Tyr503A hydrogen bond donor
Tyr108A hydrogen bond donor
Asp170A hydrogen bond donor
His422A covalently attached, activator, increase redox potential
Asp170A proton donor

Chemical Components

colligation, proton transfer, enzyme-substrate complex formation, intermediate formation

Step 5. The peroxo group deprotonates FAD, which initiates the elimination of hydrogen peroxide. It is possible that this step is catalysed by Asp170.

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Catalytic Residues Roles

Residue Roles
Arg504A hydrogen bond donor
Tyr503A hydrogen bond donor
Tyr108A hydrogen bond donor
Asp170A hydrogen bond acceptor, proton relay
His422A covalently attached, activator, increase redox potential
Asp170A proton donor, proton acceptor

Chemical Components

ingold: aromatic bimolecular elimination, proton transfer, enzyme-substrate complex cleavage, intermediate collapse, native state of cofactor regenerated, overall product formed

Step 6. Asp170 deprotonates water, which initiates a nucleophilic attack on the methoxy group, in a substitution reaction which eliminates methoxide.

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Catalytic Residues Roles

Residue Roles
Arg504A hydrogen bond donor, activator
Tyr503A hydrogen bond donor
Tyr108A hydrogen bond donor
Asp170A hydrogen bond acceptor
His422A covalently attached, increase redox potential
Asp170A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, intermediate collapse, intermediate formation, hydrolysis

Step 7. The methoxide deprotonates the alcohol group, initiating double bond rearrangement, which re-forms the phenolate moiety.

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Catalytic Residues Roles

Residue Roles
Arg504A hydrogen bond donor, electrostatic stabiliser
Tyr503A hydrogen bond donor, electrostatic stabiliser
Tyr108A hydrogen bond donor, electrostatic stabiliser
Asp170A hydrogen bond donor
His422A covalently attached, increase redox potential

Chemical Components

proton transfer, intermediate terminated, intermediate formation, overall product formed

Step 8. The pheonlate deprotonates Asp170.

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Catalytic Residues Roles

Residue Roles
His422A increase redox potential
Arg504A hydrogen bond donor, electrostatic stabiliser
Tyr503A hydrogen bond donor, electrostatic stabiliser
Tyr108A hydrogen bond donor, electrostatic stabiliser
Asp170A hydrogen bond donor
His422A covalently attached
Asp170A proton donor

Chemical Components

proton transfer, intermediate terminated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Asp170 deprotonates the 4-OH of the substrate, forming a pheonlate.

Step 2. The oxyanion collapses with double bond rearrangement, eliminating a hydride from the 1-methoxy carbon, which is added to FAD.

Step 3. The FAD undergoes double bond rearrangement which causes a single electron to be transferred to a dioxygen molecule.

Step 4. The dioxygen molecule undergoes a homolytic reaction in which it colligates to FAD, with concomitant deprotonation of Asp170.

Step 5. The peroxo group deprotonates FAD, which initiates the elimination of hydrogen peroxide. It is possible that this step is catalysed by Asp170.

Step 6. Asp170 deprotonates water, which initiates a nucleophilic attack on the methoxy group, in a substitution reaction which eliminates methoxide.

Step 7. The methoxide deprotonates the alcohol group, initiating double bond rearrangement, which re-forms the phenolate moiety.

Step 8. The pheonlate deprotonates Asp170.

Products.

Contributors

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