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PDBsum entry 1e8h
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Oxidoreductase
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PDB id
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1e8h
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structural analysis of flavinylation in vanillyl-Alcohol oxidase.
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Authors
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M.W.Fraaije,
R.H.Van den heuvel,
W.J.Van berkel,
A.Mattevi.
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Ref.
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J Biol Chem, 2000,
275,
38654-38658.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 2.
Fig. 2. The active site of the H61T ternary complex
structure showing all relevant residues, the isoalloxazine ring
of the FAD cofactor (yellow) and the bound
4-(trifluoromethyl)phenol (blue) contoured by the 2F[o]  F[c]
electron density calculated with the refined model. The picture
was generated with DINO (27).
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Figure 3.
Fig. 3. A, a section through the C  trace of
the apoenzyme subunit showing the large internal solvent-filled
cavity. For clarity, the FAD cofactor and
4-(trifluoromethyl)phenol as bound in the holo H61T ternary
complex were superimposed and shown in yellow and blue,
respectively. The surface was calculated using MSMS (28) and the
picture was generated using DINO (27). B, superposition of
active site residues in the apo (green) and the holo form
complexed with 4-(trifluoromethyl)phenol (red) structures of the
VAO H61T mutant. The figure was prepared with MOLSCRIPT ( 26).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2000,
275,
38654-38658)
copyright 2000.
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