1E8H

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

Summary for 1E8H

• Entry DOI: 10.2210/pdb1e8h/pdb
• Related: 1DZN 1E0Y 1E8F 1E8G 1QLT 1QLU
• Descriptor: VANILLYL-ALCOHOL OXIDASE, ADENOSINE-5'-DIPHOSPHATE (3 entities in total)
• Functional Keywords: oxidoreductase, flavoprotein, methanol utilization, peroxisome, flavoenzyme, oxidase, catalysis
• Biological source: PENICILLIUM SIMPLICISSIMUM
• Cellular location: Peroxisome: P56216
• Total number of polymer chains: 2
• Total formula weight: 126794.27

Authors

Mattevi, A.,Fraaije, M.W. (deposition date: 2000-09-20, release date: 2000-09-21, Last modification date: 2023-12-13)

Primary citation

Fraaije, M.W.,Van Der Heuvel, R.H.H.,Van Berkel, W.J.H.,Mattevi, A.
Structural Analysis of Flavinylation in Vanillyl-Alcohol Oxidase
J.Biol.Chem., 275:38654-, 2001

PubMed Abstract: 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.

PubMed: 10984479
DOI: 10.1074/JBC.M004753200

Experimental method

X-RAY DIFFRACTION (2.6 Å)