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 cova lently 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-C8 alpha -flavin linkage is not formed, while the enzyme is s till able to bind FAD and perform catalysis. The H61T mutant displays a sim ilar affinity for FAD and ADP (K-d = 1.8 and 2.1 muM, respectively) but doe s not interact with FMN. H61T is about 10-fold less active with 4-(methoxym ethyl)phenol) (k(cat) = 0.24 s(-1), K-m = 40 muM) than the wild-type enzyme . The crystal structures of both the hole and apo form of H61T are highly s imilar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These res ults show that covalent flavinylation is an autocatalytical process in whic h His-BI plays a crucial role by activating His-422. Furthermore, our studi es clearly demonstrate that in VAO, the FAD binds via a typical lock-and-ke y approach to a preorganized binding site.