Journal
FEBS JOURNAL
Volume 275, Issue 20, Pages 5191-5200Publisher
WILEY-BLACKWELL
DOI: 10.1111/j.1742-4658.2008.06649.x
Keywords
covalent flavinylation; FAD; post-translational modification; tandem ESI-MS; vanilly-lalcohol oxidase
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Funding
- Dutch Technology Foundation STW
- Applied Science Division of NWO
- Ministry of Economic Affairs
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Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8 alpha-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 mu M, 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.
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