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 3 Thr). In the mutant enzyme the covalent His-C8␣-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 M, 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 M) 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.A substantial part of all biological reactions rely on the action of cofactor-dependent enzymes. By adding the functionality of a cofactor to the protein matrix, enzymes have found a way of expanding their catalytic power that has led to an enormous collection of different biocatalysts. For the various types of cofactors observed in nature, different molecular approaches of cofactor binding and anchoring have been identified. Flavoproteins represent one of the major groups of cofactordependent enzymes (1, 2). However, data on the molecular process of FAD or FMN binding are scarce, while only a limited number of flavoprotein structures in the apo form are known (3-5). A striking example of the importance of efficient FAD binding was recently shown for methylenetetrahydrofolate reductase (6). A commonly found point mutation in the corresponding human gene results in a decreased cofactor affinity, which can lead to severe inborn health problems e.g. neuraltube defects and Down syndrome. For a better understanding of the process of flavin binding, we have started a study on the process of flavinylation in vanillyl-alcohol oxidase (VAO). 1 VAO (EC 1.1.3.38) is a FAD-dependent enzyme capable of oxidizing a wide range of phenolic substrates providing the fungus Penicillium simplicissimum with a tool for metabolizing aromatic compounds (7). The structure of VAO encompasses 560 residues and a fully buried covalently bound FAD cofactor ( Fig. 1) (8). The VAO monomer consists of two ␣/ domains: residues 1-270 and 500 -560 form a FAD binding domain, while the intervening residues form the so-called cap domain. The active site is located in the core of the protein, at the interface of the two domains with the flavin being covalently linked to His-422 of the cap domain. From a sequence alignment study it was recognized that VAO is a representative of a novel flavoprotein family, whose me...
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