Cofactor-dependent assembly of the flavoenzyme vanillyl-alcohol oxidase.
The oligomerization of the flavoprotein vanillyl-alcohol oxidase (VAO) and its site-directed mutant H61T was studied by mass spectrometry. Native VAO has a covalently bound FAD and forms primarily octameric assemblies of 507 kDa. H61T is purified as a FAD-free apoprotein and mainly exists as a dimeric species of 126 kDa. Binding of FAD to apoH61T rapidly restores enzyme activity and induces octamerization, although association of H61T dimers seems not to be crucial for enzyme activity. Reconstitution of H61T with the cofactor analog 5'-ADP also promotes octamerization. FMN on the other hand, interacts with apoH61T without stimulating dimer association. These results are in line with observations made for several other flavoenzymes, which contain a Rossmann fold. Members of the VAO flavoprotein family do not contain a Rossmann fold but do share two conserved loops that are responsible for binding the pyrophosphate moiety of FAD. Therefore, the observed FAD-induced oligomerization might be general for this family. We speculate that upon FAD binding, small conformational changes in the ADP-binding pocket of the dimeric VAO species are transmitted to the protein surface, promoting oligomerization.
Tahallah
Nora
N
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Van Den Heuvel
Robert H H
RH
Van Den Berg
Willy A M
WA
Maier
Claudia S
CS
Van Berkel
Willem J H
WJ
Heck
Albert J R
AJ
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D019277:Entropy; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010407:Penicillium / Q000201:enzymology; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
D005182:Flavin-Adenine Dinucleotide; D000429:Alcohol Oxidoreductases; C076865:vanillyl-alcohol oxidase