1qlu: Difference between revisions
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==Overview== | ==Overview== | ||
By mutating the target residue of covalent flavinylation in, vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was, studied. Three His(422) mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state, kinetics revealed that the mutants have retained enzyme activity, although, the turnover rates have decreased by 1 order of magnitude. Stopped-flow, analysis showed that the H422A mutant is still able to form a stable, binary complex of reduced enzyme and a quinone methide product, intermediate, a crucial step during vanillyl-alcohol oxidase-mediated, catalysis. The only significant change in the catalytic cycle of the H422A, mutant is a marked decrease in reduction rate. Redox potentials of both, wild type and ... | By mutating the target residue of covalent flavinylation in, vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was, studied. Three His(422) mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state, kinetics revealed that the mutants have retained enzyme activity, although, the turnover rates have decreased by 1 order of magnitude. Stopped-flow, analysis showed that the H422A mutant is still able to form a stable, binary complex of reduced enzyme and a quinone methide product, intermediate, a crucial step during vanillyl-alcohol oxidase-mediated, catalysis. The only significant change in the catalytic cycle of the H422A, mutant is a marked decrease in reduction rate. Redox potentials of both, wild type and H422A vanillyl-alcohol oxidase have been determined. During, reduction of H422A, a large portion of the neutral flavin semiquinone is, observed. Using suitable reference dyes, the redox potentials for the two, one-electron couples have been determined: -17 and -113 mV. Reduction of, wild type enzyme did not result in any formation of flavin semiquinone and, revealed a remarkably high redox potential of +55 mV. The marked decrease, in redox potential caused by the missing covalent histidyl-FAD bond is, reflected in the reduced rate of substrate-mediated flavin reduction, limiting the turnover rate. Elucidation of the crystal structure of the, H422A mutant established that deletion of the histidyl-FAD bond did not, result in any significant structural changes. These results clearly, indicate that covalent interaction of the isoalloxazine ring with the, protein moiety can markedly increase the redox potential of the flavin, cofactor, thereby facilitating redox catalysis. Thus, formation of a, histidyl-FAD bond in specific flavoenzymes might have evolved as a way to, contribute to the enhancement of their oxidative power. | ||
==About this Structure== | ==About this Structure== | ||
1QLU is a | 1QLU is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Penicillium_simplicissimum Penicillium simplicissimum] with FAD and EUG as [http://en.wikipedia.org/wiki/ligands ligands]. 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] Structure known Active Sites: FA1 and FA2. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1QLU OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: peroxisome]] | [[Category: peroxisome]] | ||
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 5 14:39:41 2007'' | ||
Revision as of 15:34, 5 November 2007
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STRUCTURE OF THE H422A MUTANT VANILLYL-ALCOHOL OXIDASE IN COMPLEX WITH ISOEUGENOL
OverviewOverview
By mutating the target residue of covalent flavinylation in, vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was, studied. Three His(422) mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state, kinetics revealed that the mutants have retained enzyme activity, although, the turnover rates have decreased by 1 order of magnitude. Stopped-flow, analysis showed that the H422A mutant is still able to form a stable, binary complex of reduced enzyme and a quinone methide product, intermediate, a crucial step during vanillyl-alcohol oxidase-mediated, catalysis. The only significant change in the catalytic cycle of the H422A, mutant is a marked decrease in reduction rate. Redox potentials of both, wild type and H422A vanillyl-alcohol oxidase have been determined. During, reduction of H422A, a large portion of the neutral flavin semiquinone is, observed. Using suitable reference dyes, the redox potentials for the two, one-electron couples have been determined: -17 and -113 mV. Reduction of, wild type enzyme did not result in any formation of flavin semiquinone and, revealed a remarkably high redox potential of +55 mV. The marked decrease, in redox potential caused by the missing covalent histidyl-FAD bond is, reflected in the reduced rate of substrate-mediated flavin reduction, limiting the turnover rate. Elucidation of the crystal structure of the, H422A mutant established that deletion of the histidyl-FAD bond did not, result in any significant structural changes. These results clearly, indicate that covalent interaction of the isoalloxazine ring with the, protein moiety can markedly increase the redox potential of the flavin, cofactor, thereby facilitating redox catalysis. Thus, formation of a, histidyl-FAD bond in specific flavoenzymes might have evolved as a way to, contribute to the enhancement of their oxidative power.
About this StructureAbout this Structure
1QLU is a Single protein structure of sequence from Penicillium simplicissimum with FAD and EUG as ligands. Active as Aryl-alcohol oxidase, with EC number 1.1.3.7 Structure known Active Sites: FA1 and FA2. Full crystallographic information is available from OCA.
ReferenceReference
Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase., Fraaije MW, van den Heuvel RH, van Berkel WJ, Mattevi A, J Biol Chem. 1999 Dec 10;274(50):35514-20. PMID:10585424
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