Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process

Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8α-histidyl bound FAD, which represents the most frequently encountered covalent flavin–protein linkage. To elucidate the mechanism by which VAO covalently incorporates the FAD cofactor, apo VAO was produced by using a riboflavin auxotrophic Escherichia coli strain. Incubation of apo VAO with FAD resulted in full restoration of enzyme activity. The rate of activity restoration was dependent on FAD concentration, displaying a hyperbolic relationship (KFAD = 2.3 µm, kactivation = 0.13 min-1). The time-dependent increase in enzyme activity was accompanied by full covalent incorporation of FAD, as determined by SDS⁄PAGE and ESI-MS analysis. The results obtained show that formation of the covalent flavin–protein bond is an autocatalytic process, which proceeds via a reduced flavin intermediate. Furthermore, ESI-MS experiments revealed that, although apo VAO mainly exists as monomers and dimers, FAD binding promotes the formation of VAO dimers and octamers. Tandem ESI-MS experiments revealed that octamerization is not dependent on full covalent flavinylation.