Covalent Flavinylation Is Essential for Efficient Redox Catalysis in Vanillyl-alcohol Oxidase

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 vanillylalcohol 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.Until now, several hundred flavin-containing enzymes have been described. Most of these enzymes contain a dissociable FAD or FMN cofactor. However, it has been shown that in several cases the flavin is covalently linked to an amino acid of the polypeptide chain. In fact, in humans 10% of the cellular FAD is covalently bound to enzymes like e.g. succinate dehydrogenase and monoamine oxidase (1). Within the group of covalent flavoproteins, five different types of covalent flavinylation have been identified. Except for a few examples of cysteinyl-or tyrosyl-linked flavins, tethering to a histidine is by far the most favored binding mode, since it has been observed in about 20 isolated flavoenzymes (for a recent review, see Ref. 2).Although the first covalent flavoprotein, succinate dehydrogenase, was already identified in 1955 (3), the rationale for covalent flavinylation is still unresolved. Only recently, a clear influence of the covalent bond on the reactivity of the cofactor has been observed in trimethylamine dehydrogenase. Unlike the wild type enzyme, mutants of trimethylamine dehydrogenase containing dissociable FMN (4, 5) are inactivated b...

Inversion of stereospecificity of vanillyl-alcohol oxidase

“…Previous studies have indicated that the covalent interaction between His-422 and the isoalloxazine ring markedly increases the redox potential of the flavin cofactor (10). The redox potentials of D170A͞T457E and D170S͞T457E were only slightly lower than that of wild-type VAO, supporting the proposal that an acidic residue in the active site cavity is required for maintaining the high redox potential (11).…”

“…The inhibitor was clearly visible in the electron density map of one of the two crystallographically independent subunits (subunit B). Its orientation in the proximity of the flavin ring was nearly identical to the orientation in several previously determined enzymeinhibitor complex structures (8,10,11). Superposition of the structures of trifluoromethylphenol-complexed D170S͞T457E and isoeugenol-complexed wild-type VAO (8) showed that the two amino acid replacements did not cause any significant conformational change, which is reflected in a rms deviation for all C␣-atoms of only 0.23 Å.…”

“…SDS͞PAGE was carried out in 12.5% slab gels essentially as reported earlier (21,22). The redox potentials of the VAO mutants were determined by the xanthine͞xanthine oxidase method (23), essentially as described before (10,11).…”

“…All present evidence suggests that the covalent anchoring of the flavin is a selfcatalytic process (9). From the properties of His-422 variants, it was established that the covalent interdomain interaction with His-422 serves to increase the flavin redox potential, an essential feature for efficient redox catalysis (10). Another important residue in the enzyme active site is Asp-170.…”

Vitamin C biosynthesis in trypanosomes: A role for the glycosome

“…This is similar to plant GALDHs, where leucine is found at this site, leading to the suggestion that plant enzymes may interact noncovalently with FAD (28,29). In the related flavoprotein vanillyl-alcohol oxidase (VAO), the equivalent histidine facilitates flavinylation at a second histidine residue near the C terminus (30). This second histidine is conserved at a corresponding position in all GLO͞ALO͞GALDH sequences reported (residue 462 in TbALO) (Fig.…”