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