4-(Methoxymethyl)phenol

Structural analysis of flavinylation in vanillyl-alcohol oxidase.[Pubmed:10984479]

J Biol Chem. 2000 Dec 8;275(49):38654-8.

Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8alpha-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K(d) = 1.8 and 2.1 microm, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(Methoxymethyl)phenol) (k(cat) = 0.24 s(-)(1), K(m) = 40 microm) than the wild-type enzyme. The crystal structures of both the holo and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-61 plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site.

Asp-170 is crucial for the redox properties of vanillyl-alcohol oxidase.[Pubmed:10809721]

J Biol Chem. 2000 May 19;275(20):14799-808.

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.

Subcellular localization of vanillyl-alcohol oxidase in Penicillium simplicissimum.[Pubmed:9475171]

FEBS Lett. 1998 Jan 23;422(1):65-8.

Growth of Penicillium simplicissimum on anisyl alcohol, veratryl alcohol or 4-(Methoxymethyl)phenol, is associated with the synthesis of relatively large amounts of the hydrogen peroxide producing flavoprotein vanillyl-alcohol oxidase (VAO). Immunocytochemistry revealed that the enzyme has a dual location namely in peroxisomes and in the cytosol. The C-terminus of the primary structure of VAO displays a WKL-COOH sequence which might function as a peroxisomal targeting signal type 1 (PTS1). As VAO activity results in production of hydrogen peroxide also the subcellular location of a recently characterized co-inducible catalase-peroxidase was studied. As VAO, this hydroperoxidase is also distributed throughout the cytosol and peroxisomes.

Catalytic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol by vanillyl-alcohol oxidase. Evidence for formation of a p-quinone methide intermediate.[Pubmed:9218444]

J Biol Chem. 1997 Jul 18;272(29):18111-6.

The catalytic mechanism for the oxidative demethylation of 4-(Methoxymethyl)phenol by the covalent flavoprotein vanillyl-alcohol oxidase was studied. Using H218O, it was found that the carbonylic oxygen atom from the product 4-hydroxybenzaldehyde originates from a water molecule. Oxidation of vanillyl alcohol did not result in any incorporation of 18O. Enzyme-monitored turnover experiments revealed that for both substrates a process involving flavin reduction is rate determining. During anaerobic reduction of vanillyl-alcohol oxidase by 4-(Methoxymethyl)phenol, a relatively stable spectral intermediate is formed. Deconvolution of its spectral characteristics showed a typical pH-independent absorption maximum at 364 nm (epsilon364 nm = 46 mM-1 cm-1). A similar transient species was observed upon anaerobic reduction by vanillyl alcohol. The rate of flavin reduction and synchronous intermediate formation by 4-(Methoxymethyl)phenol is 3.3 s-1 and is fast enough to account for turnover (3.1 s-1). The anaerobic decay of the intermediate was too slow (0.01 s-1) to be of catalytical relevance. The reduced binary complex is rapidly reoxidized (1.5 x 10(5) M-1 s-1) and is accompanied with formation and release of product. Oxidation of free-reduced enzyme is an even faster process (3.1 x 10(5) M-1 s-1). The kinetic data for the oxidative demethylation of 4-(Methoxymethyl)phenol are in accordance with a ternary complex mechanism in which the reduction rate is rate-limiting. It is proposed that, upon reduction, a binary complex is produced composed of the p-quinone methide of 4-(Methoxymethyl)phenol and reduced enzyme.

Enigmatic Gratuitous Induction of the Covalent Flavoprotein Vanillyl-Alcohol Oxidase in Penicillium simplicissimum.[Pubmed:16535508]

Appl Environ Microbiol. 1997 Feb;63(2):435-9.

When Penicillium simplicissimum is grown on veratryl alcohol, anisyl alcohol, or 4-(Methoxymethyl)phenol, an intracellular covalent flavin-containing vanillyl-alcohol oxidase is induced. The induction is highest (up to 5% of total protein) during the growth phase. In addition to vanillyl-alcohol oxidase, an intracellular catalase-peroxidase is induced. Induction of vanillyl-alcohol oxidase in P. simplicissimum is prevented by the addition of isoeugenol to veratryl alcohol-containing media, but growth is unaffected. The inhibitory effect of isoeugenol on induction is not observed when anisyl alcohol or 4-(Methoxymethyl)phenol is used as the growth substrate. Based on the induction experiments and the degradation pathways for veratryl and anisyl alcohol, we propose that induction of vanillyl-alcohol oxidase is superfluous when P. simplicissimum is grown on these aromatic alcohols. However, the enzyme plays an essential role in the degradation of the methyl ether of p-cresol, 4-(Methoxymethyl)phenol.

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