Structural analysis of flavinylation in vanillyl-alcohol oxidase

M.W. Fraaije, R.H.H. van den Heuvel, W.J.H. van Berkel, A. Mattevi

Research output: Contribution to journalArticleAcademicpeer-review

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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-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.
Original languageEnglish
Pages (from-to)38654-38658
JournalJournal of Biological Chemistry
Volume275
DOIs
Publication statusPublished - 2000

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vanillyl-alcohol oxidase
Flavin-Adenine Dinucleotide
Structural analysis
Enzymes
Flavin Mononucleotide
Flavoproteins
Apoproteins
Catalysis
Point Mutation
Adenosine Diphosphate
Oxidoreductases
Crystal structure
Binding Sites

Cite this

Fraaije, M.W. ; van den Heuvel, R.H.H. ; van Berkel, W.J.H. ; Mattevi, A. / Structural analysis of flavinylation in vanillyl-alcohol oxidase. In: Journal of Biological Chemistry. 2000 ; Vol. 275. pp. 38654-38658.
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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-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.",
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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 journalArticleAcademicpeer-review

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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

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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.

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