Covalent flavinylation enhances the oxidative power of vanillyl-alcohol oxidase

Vanillyl-alcohol oxidase (VAO) from Penicillium simplicissimum is an inducible flavoprotein that is active with a wide range of phenolic compounds. The enzyme is the prototype of a newly recognized family of structurally related oxidoreductases, whose members share a conserved FAD-binding domain. The flavin cofactor in VAO is covalently linked to His422 of the cap domain. Studies from His422 variants revealed that deletion of the histidyl–flavin bond does not result in any significant structural change. However, the covalent interaction increases the redox potential of the flavin, facilitating substrate oxidation. His61, located in the FAD-binding domain, is involved in the autocatalytic process of covalent flavinylation. This could be nicely demonstrated by creating the H61T mutant enzyme which binds the flavin in a non-covalently mode. Similar to the noncovalent His422 variants, H61T is 10-fold less active than wild-type VAO. From this and the similar crystal structures of apo and holo H61T it is concluded that the FAD binds to a preorganized binding site where His61 activates His422 for autocatalytic flavinylation.