title: Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process.
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.
authors.affiliation: Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Jin
authors.forename: Hortense
authors.initials: H
authors.lastname: Mazon
authors.forename: Robert H H
authors.initials: RH
authors.lastname: van den Heuvel
authors.forename: Albert J
authors.initials: AJ
authors.lastname: Heck
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D002384:Catalysis; D019281:Dimerization; D029968:Escherichia coli Proteins; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding
chemical_list: D029968:Escherichia coli Proteins; D005182:Flavin-Adenine Dinucleotide; D000429:Alcohol Oxidoreductases; C076865:vanillyl-alcohol oxidase