1dzn
From Proteopedia
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'''ASP170SER MUTANT OF VANILLYL-ALCOHOL OXIDASE''' | '''ASP170SER MUTANT OF VANILLYL-ALCOHOL OXIDASE''' | ||
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[[Category: Heuvel, R H.H Van Den.]] | [[Category: Heuvel, R H.H Van Den.]] | ||
[[Category: Mattevi, A.]] | [[Category: Mattevi, A.]] | ||
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| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 14:28:49 2008'' | |
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Revision as of 11:28, 2 May 2008
ASP170SER MUTANT OF VANILLYL-ALCOHOL OXIDASE
Overview
Vanillyl-alcohol oxidase is a flavoprotein containing a covalent flavin that catalyzes the oxidation of 4-(methoxymethyl)phenol to 4-hydroxybenzaldehyde. The reaction proceeds through the formation of a p-quinone methide intermediate, after which, water addition takes place. Asp-170, located near the N5-atom of the flavin, has been proposed to act as an active site base. To test this hypothesis, we have addressed the properties of D170E, D170S, D170A, and D170N variants. Spectral and fluorescence analysis, together with the crystal structure of D170S, suggests that the Asp-170 replacements do not induce major structural changes. However, in D170A and D170N, 50 and 100%, respectively, of the flavin is non-covalently bound. Kinetic characterization of the vanillyl-alcohol oxidase variants revealed that Asp-170 is required for catalysis. D170E is 50-fold less active, and the other Asp-170 variants are about 10(3)-fold less active than wild type enzyme. Impaired catalysis of the Asp-170 variants is caused by slow flavin reduction. Furthermore, the mutant proteins have lost the capability of forming a stable complex between reduced enzyme and the p-quinone methide intermediate. The redox midpoint potentials in D170E (+6 mV) and D170S (-91 mV) are considerably decreased compared with wild type vanillyl-alcohol oxidase (+55 mV). This supports the idea that Asp-170 interacts with the protonated N5-atom of the reduced cofactor, thus increasing the FAD redox potential. Taken together, we conclude that Asp-170 is involved in the process of autocatalytic flavinylation and is crucial for efficient redox catalysis.
About this Structure
1DZN is a Single protein structure of sequence from Penicillium simplicissimum. Full crystallographic information is available from OCA.
Reference
Asp-170 is crucial for the redox properties of vanillyl-alcohol oxidase., van den Heuvel RH, Fraaije MW, Mattevi A, van Berkel WJ, J Biol Chem. 2000 May 19;275(20):14799-808. PMID:10809721 Page seeded by OCA on Fri May 2 14:28:49 2008
