Structural analysis of flavinylation in vanillyl-alcohol oxidase.
Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8alpha-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K(d) = 1.8 and 2.1 microm, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-)(1), K(m) = 40 microm) than the wild-type enzyme. The crystal structures of both the holo and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-61 plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site.
Fraaije
M W
MW
Department of Biomolecular Sciences, Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. m.w.fraaije@chem.rug.nl
van Den Heuvel
R H
RH
van Berkel
W J
WJ
Mattevi
A
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D001051:Apoenzymes / Q000737:chemistry; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D010407:Penicillium / Q000201:enzymology; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001051:Apoenzymes; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; D000244:Adenosine Diphosphate; D005486:Flavin Mononucleotide; D000429:Alcohol Oxidoreductases; C076865:vanillyl-alcohol oxidase