Related Experiment Video
Updated: Jul 1, 2026

HKUST-1 as a Heterogeneous Catalyst for the Synthesis of Vanillin
Published on: July 23, 2016
Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process
Jianfeng Jin1, Hortense Mazon, Robert H H van den Heuvel
1Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
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.
Related Concept Videos
Oxidation of Alcohols
The process of oxidation in a chemical reaction is observed in any of the three forms:
Oxidation of Alkenes: Syn Dihydroxylation with Potassium Permanganate
Vicinal Diols via Reductive Coupling of Aldehydes or Ketones: Pinacol Coupling Overview
Oxidations of Aldehydes and Ketones to Carboxylic Acids
Aldehydes readily undergo oxidation in strong oxidizing agents such as potassium permanganate and chromic acid. The oxidation can also be carried out using mild oxidizing agents such as silver oxide. In fact, aldehydes can be easily oxidized...
Oxidation of Alkenes: Syn Dihydroxylation with Osmium Tetraoxide
Oxidation of Phenols to Quinones
o-hydroxy phenols are oxidized to o-quinones and p-hydroxy phenols to p-quinones. Such redox reactions involve the transfer of two electrons and two protons. The reversible redox property is crucial in...

