Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process.
- Authors
- Type
- Published Article
- Journal
- FEBS Journal
- Publisher
- Wiley (Blackwell Publishing)
- Publication Date
- Oct 01, 2008
- Volume
- 275
- Issue
- 20
- Pages
- 5191–5200
- Identifiers
- DOI: 10.1111/j.1742-4658.2008.06649.x
- PMID: 18793324
- Source
- Medline
- License
- Unknown
Abstract
Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8alpha-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 (K(FAD )= 2.3 microM, k(activation) = 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.