Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 38654-38658 |
| Journal | Journal of Biological Chemistry |
| Volume | 275 |
| DOIs | |
| Publication status | Published - 2000 |
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Structural analysis of flavinylation in vanillyl-alcohol oxidase. / Fraaije, M.W.; van den Heuvel, R.H.H.; van Berkel, W.J.H.; Mattevi, A.
In: Journal of Biological Chemistry, Vol. 275, 2000, p. 38654-38658.Research output: Contribution to journal › Article › Academic › peer-review
TY - JOUR
T1 - Structural analysis of flavinylation in vanillyl-alcohol oxidase
AU - Fraaije, M.W.
AU - van den Heuvel, R.H.H.
AU - van Berkel, W.J.H.
AU - Mattevi, A.
PY - 2000
Y1 - 2000
N2 - 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-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 (Kd = 1.8 and 2.1 ?M, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (kcat = 0.24 s1, Km = 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.
AB - 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-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 (Kd = 1.8 and 2.1 ?M, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (kcat = 0.24 s1, Km = 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.
U2 - 10.1074/jbc.M004753200
DO - 10.1074/jbc.M004753200
M3 - Article
VL - 275
SP - 38654
EP - 38658
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
ER -