1e8h

From Proteopedia

Revision as of 14:02, 5 November 2007

STRUCTURE OF THE H61T MUTANT OF THE FLAVOENZYME VANILLYL-ALCOHOL OXIDASE IN THE APO FORM COMPLEXED BY ADP

Overview

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.

About this Structure

1E8H is a Single protein structure of sequence from Penicillium simplicissimum with ADP as ligand. Active as Aryl-alcohol oxidase, with EC number 1.1.3.7 Structure known Active Sites: AC1 and AC2. Full crystallographic information is available from OCA.

Reference

Structural analysis of flavinylation in vanillyl-alcohol oxidase., Fraaije MW, van Den Heuvel RH, van Berkel WJ, Mattevi A, J Biol Chem. 2000 Dec 8;275(49):38654-8. PMID:10984479

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