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Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process.

Authors
  • Jin, Jianfeng
  • Mazon, Hortense
  • van den Heuvel, Robert H H
  • Heck, Albert J
  • Janssen, Dick B
  • Fraaije, Marco W
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.

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