Vanillyl-alcohol oxidase
- UniProt: P56216
- CAS Number: 143929-24-2
Vanillyl alcohol oxidase ( VAO, EC 1.1.3.38 ) is an enzyme in bacteria and fungi, which catalyzes the oxidation of various phenolic compounds, using oxygen (O2). This produces hydrogen peroxide ( H2O2). For the reaction, as a cofactor Flavinmolekül, FAD, are required. VSOs are localized in the peroxisomes and belong to a special type of aryl alcohol oxidases in, they are for the lignin by microorganisms of importance.
1992 the first time the vanillyl alcohol oxidase was isolated from the ascomycete Penicillium simplicissimum and characterized in 1997 and its structure clarified. VAO is highly expressed in this fungus when it responds to veratryl alcohol, anisyl alcohol or 4 - grows ( methoxymethyl ) phenol.
The article refers mostly data on the enzyme from P. simplicissimum.
Classification
The intracellular vanillyl-alcohol oxidase is one of the aryl alcohol oxidases. These include, in turn, to the widespread group of oxidoreductases, which all have a conserved FAD - binding domain. By amino acid sequence comparisons show that the vanillyl alcohol oxidase forms a separate class that the VAO Flavoproteinfamilie. To this family include, for example eugenol oxidase, Chloesteroloxidase ( EC 1.1.3.6 ) and the alditol oxidase.
Catalyzed reaction
VAO catalyzes the oxidation of various phenolic compounds, especially numerous 4- alkylphenols. Originally in - vitro data have shown the oxidation of vanillyl alcohol to vanillin:
The physiological significance of this reaction is not entirely known. Vanillyl so is not an intermediate in the degradation of veratryl alcohol, which induces the production of VAO in P. simplicissimum.
Further VAO can oxidatively Methoxykresol p- (4 - ( methoxymethyl ) phenol ) demethylating to 4 -hydroxybenzaldehyde. This produces methanol. In the reaction, water is used as the nucleophilic agent and forms the aldehyde group in the newly formed 4- hydroxybenzaldehyde. During catalysis, FAD is first reduced and then reoxidized by molecular oxygen. This produces hydrogen peroxide. p- Methoxykresol is a physiological substrate.
Also deamination vanillylamine to vanillin was detected. The conversion of eugenol to coniferyl alcohol is also catalyzed by VAO, which generally represents an oxidative hydration of 4- allyl phenols. This demonstrates the highest enzymatic activity: eugenol and chavicol are the best substrates for VAO.
VAO from P. simplicissimum shows - in vitro - an optimum pH from 9 to 10.5 and a temperature optimum at 38 ° C.
Structure
The VAO from P. simplicissimum is an octamer of the same subunits. This form tetramers of dimers the forest. The octamer is approximately 500 kDa heavy. In the presence of chaotropic agents, the octamer is divided into dimers. The dimers also exhibit catalytic activity. The monomer is 560 amino acids and 64 kDa in size and consists of two domains, an FAD - binding domain and a cap domain. The latter is necessary for the substrate binding. Each monomer contributes as a bound cofactor Flavinmolekül. This is covalently linked via the isoalloxazine ring of an L- histidine ( His422 ).
The catalytic center
The catalytic center of the enzyme forms a water- inaccessible cave with about 200 Å3 volume. It is surrounded by hydrophobic and aromatic amino acids.
FAD
VAO is the first enzyme of known structure, wherein the C8 carbon atom of a Flavinmoleküls is linked covalently to a Nε2 atom of histidine 422 ( see figure). The planarity of the isoalloxazin ring is not changed. The isoalloxazine ring also forms several hydrogen bonds to the apoprotein.
The prosthetic group is important for enzyme activity. It is believed that the covalent bond to the protein significantly increases the redox potential of the co-factor for catalysis. This is followed is also an amino acid of the protein, L- aspartate 170, involved. Probably FAD is linked post-translationally to the apoprotein in an autocatalytic process.
Inhibitors
For VAO a number of inhibitors have been identified. Isoeugenol, 2-nitro -p-cresol, p- cresol, coniferyl alcohol ( product of the reaction with eugenol ), and cinnamyl alcohol inhibit the enzyme in a competitive manner.
Importance
Demethylation of 4 - ( methoxymethyl ) phenol, in the ascomycete of physiological importance, as VAO initiates the pathway of this compound. Derivatives of this compound are quite degradation products of lignin. As a result, VAO can be used for the metabolism of these substrates and play a role in lignin degradation.
The enzyme catalyzes (in vitro) of the product formation of two monolignols, coniferyl alcohol and coumaryl alcohol. Monolignols are generated mainly from plants in the wake of Ligninbildung. The third Monolignol, sinapyl alcohol, but can not be produced by VAO.
In contrast to the extracellular aryl alcohol oxidases many stand mushrooms the substrate specificity of the VAO is much narrower. Through its range of the enzyme catalysis for the chemical industry could be found as a biocatalyst feeder.