1QLT

STRUCTURE OF THE H422A MUTANT OF THE FLAVOENZYME VANILLYL-ALCOHOL OXIDASE

Summary for 1QLT

• Entry DOI: 10.2210/pdb1qlt/pdb
• Related: 1AHU 1AHV 1AHZ 1QLU 1VAO 2VAO
• Descriptor: VANILLYL-ALCOHOL OXIDASE, FLAVIN-ADENINE DINUCLEOTIDE, ACETATE ION, ... (4 entities in total)
• Functional Keywords: oxidoreductase, flavoprotein, methanol utilization, peroxisome, flavoenzyme, oxidase, catalysis
• Biological source: PENICILLIUM SIMPLICISSIMUM
• Total number of polymer chains: 2
• Total formula weight: 127569.01

Authors

Mattevi, A.,Fraaije, M. (deposition date: 1999-09-16, release date: 1999-09-20, Last modification date: 2024-05-08)

Primary citation

Fraaije, M.W.,van den Heuvel, R.H.,van Berkel, W.J.,Mattevi, A.
Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase.
J.Biol.Chem., 274:35514-35520, 1999

PubMed Abstract: 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.

PubMed: 10585424
DOI: 10.1074/jbc.274.50.35514

Experimental method

X-RAY DIFFRACTION (2.2 Å)