Crystallization and preliminary x-ray analysis of the flavoenzyme vanillyl-alcohol oxidase fromPenicillium Simplicissimum

Mattevi, Andrea; Fraaije, Marco W.; Coda, Alessandro; Berkel, Willem J.H. van

doi:10.1002/(sici)1097-0134(199704)27:4<601::aid-prot12>3.0.co;2-o

Cited by 20 publications

(18 citation statements)

References 12 publications

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“…VAO represents a novel type of flavoprotein oxidase [2] and its X-ray structure is presently determined by the method of multiple isomorphous replacement [3]. In soaking experiments with mercury compounds we noticed that the VAO crystals are sensitive towards structural changes, influencing the crystal packing.…”

Section: Discussionmentioning

confidence: 99%

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Mercuration of vanillyl‐alcohol oxidase from Penicillium simplicissimum generates inactive dimers

Fraaije¹,

Mattevi

Berkel³

1997

FEBS Letters

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Vanillyl-alcohol oxidase (EC 1.1.3.7) from Penicillium simplicissimum was modified with p-mercuribenzoate. One cysteine residue reacts rapidly without loss of enzyme activity. Three sulfhydryl groups then react in an 'all or none process' involving enzyme inactivation and dissociation of the octamer into dimers. The inactivation reaction is slowed down in the presence of the competitive inhibitor isoeugenol and fully reversible by treatment of the modified enzyme with dithiothreitol. Vanillyl-alcohol oxidase is more rapidly inactivated at low enzyme concentrations and protected from mercuration by antichaotropic salts. It is proposed that subunit dissociation accounts for the observed sensitivity of vanillyl-alcohol oxidase crystals towards mercury compounds.

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Section: Discussionmentioning

confidence: 99%

“…This indicates that at least one of the cysteine residues is located near the dimer-dimer interface. Because VAO crystals grow in low salt [3] it is proposed that subunit dissociation accounts for the observed sensitivity of VAO crystals towards mercury compounds.…”

Section: Discussionmentioning

confidence: 99%

Mercuration of vanillyl‐alcohol oxidase from Penicillium simplicissimum generates inactive dimers

Fraaije¹,

Mattevi

Berkel³

1997

FEBS Letters

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“…Crystallization, Data Collection, and Processing-Bright yellow crystals of all His 422 mutants were obtained using the hanging drop vapor diffusion method at similar conditions (5% (w/v) polyethylene glycol 4000, 100 mM sodium acetate/HCl, pH 5.1) that were used for crystallizing wild type VAO (32). Crystallization of H422A resulted in the most regularly shaped crystals.…”

Section: Methodsmentioning

confidence: 99%

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

Fraaije¹,

Heuvel²,

Berkel³

et al. 1999

Journal of Biological Chemistry

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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...

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“…The oxidase has been crystallized (Croteau & Vrielink, 1996). Currently, the crystal structures are known for PCMH (Sa-0-tyrosyl-FAD), TMADH (6-S-cysteinyl-FMN; see below), and flavocytochrome c552 (8a-S-cysteinyl-FAD), and the analysis of the crystal structure of vanillyl oxidase (Sa-N1-histidyl-FAD) is in progress (Mattevi et al, 1997). Hopefully, the structure of the cholesterol oxidase will soon be solved.…”

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Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs

1998

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The first identified covalent flavoprotein, a component of mammalian succinate dehydrogenase, was reported 42 years ago. Since that time, more than 20 covalent flavoenzymes have been described, each possessing one of five modes of FAD or FMN linkage to protein. Despite the early identification of covalent flavoproteins, the mechanisms of covalent bond formation and the roles of the covalent links are only recently being appreciated. The main focus of this review is, therefore, one of mechanism and function, in addition to surveying the types of linkage observed and the methods employed for their identification. Case studies are presented for a variety of covalent flavoenzymes, from which general findings are beginning to emerge.Keywords: covalent flavoproteins; flavin adenine dinucleotide; flavin mononucleotide; flavinylation; redox cofactors Cofactors are recruited by protein molecules to extend the chemistry available within the active sites of enzymes. The chemistries displayed are variable and the range of protein-specific cofactors available bears testimony to the types of biological reaction achieved by many enzyme molecules. Cofactors may be present in the freely dissociable form, but others, such as lipoic acid and biotin, are permanently linked to the host protein. Other cofactors (e.g., heme and pyridoxyl phosphate) fall into both categories in that they can be attached covalently to the protein or operate in the freely dissociable form. The covalent addition of specific cofactors to unique residues within a polypeptide chain is a fundamental problem in studies of biological molecular recognition. In many cases, this specificity is purchased at the expense of recruiting modification enzymes that direct the covalent addition of a cofactor to the host protein. For example, the additions of biotin and lipoic acid to the €-amino groups of specific Lys residues in carboxyltransferases and the 2-oxoacid dehydrogenases are directed by a biotin holoenzyme synthase (Sweetman et al., 1985;Takai et al., 1987) and Reprint requests to: N.S. Scmtton, Department of Biochemistry, University of Leicester, Adrian Building, University Road, Leicester LE1 7RH UK; e-mail: nss4@le.ac.uk; or W.S. McIntire, Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121; e-mail: wsm@itsa.ucsf.edu.lipoate-protein ligases (Brookfield et al., 1991; Moms et al., 1994), respectively. In a similar fashion, a heme cytochrome c lyase (Steiner et al., 1996) is responsible for the covalent addition of heme to two Cys residues via thioether linkages. In some enzymes, an existing side chain is modified to yield what is in effect a covalently attached cofactor, although no pre-existing cofactor molecule is incorporated into the enzyme. Several examples of this type of modification are provided in the next paragraph.For histidine decarboxylase of Lactobacillus 30A (Recsei & Snell, 1984), a pyruvoyl group is generated from an existing residue, Ser 82. The enzyme is synthesized as a proenzym...

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Crystallization and preliminary x-ray analysis of the flavoenzyme vanillyl-alcohol oxidase fromPenicillium Simplicissimum

Cited by 20 publications

References 12 publications

Mercuration of vanillyl‐alcohol oxidase from Penicillium simplicissimum generates inactive dimers

Mercuration of vanillyl‐alcohol oxidase from Penicillium simplicissimum generates inactive dimers

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

Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs

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