1e8f
From Proteopedia
(New page: 200px<br /><applet load="1e8f" size="450" color="white" frame="true" align="right" spinBox="true" caption="1e8f, resolution 2.9Å" /> '''STRUCTURE OF THE H61T...) |
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| - | [[Image:1e8f.gif|left|200px]]<br /><applet load="1e8f" size=" | + | [[Image:1e8f.gif|left|200px]]<br /><applet load="1e8f" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1e8f, resolution 2.9Å" /> | caption="1e8f, resolution 2.9Å" /> | ||
'''STRUCTURE OF THE H61T MUTANT OF THE FLAVOENZYME VANILLYL-ALCOHOL OXIDASE IN THE APO FORM'''<br /> | '''STRUCTURE OF THE H61T MUTANT OF THE FLAVOENZYME VANILLYL-ALCOHOL OXIDASE IN THE APO FORM'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Vanillyl-alcohol oxidase (VAO) is member of a newly recognized | + | 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== | ==About this Structure== | ||
| - | 1E8F is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Penicillium_simplicissimum Penicillium simplicissimum]. Active as [http://en.wikipedia.org/wiki/Aryl-alcohol_oxidase Aryl-alcohol oxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.3.7 1.1.3.7] Full crystallographic information is available from [http:// | + | 1E8F is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Penicillium_simplicissimum Penicillium simplicissimum]. Active as [http://en.wikipedia.org/wiki/Aryl-alcohol_oxidase Aryl-alcohol oxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.3.7 1.1.3.7] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1E8F OCA]. |
==Reference== | ==Reference== | ||
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[[Category: peroxisome]] | [[Category: peroxisome]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:24:58 2008'' |
Revision as of 10:25, 21 February 2008
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STRUCTURE OF THE H61T MUTANT OF THE FLAVOENZYME VANILLYL-ALCOHOL OXIDASE IN THE APO FORM
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
1E8F is a Single protein structure of sequence from Penicillium simplicissimum. Active as Aryl-alcohol oxidase, with EC number 1.1.3.7 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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