Function, mechanism and structure of vanillyl-alcohol oxidase

Lignin is a heterogeneous aromatic polymer formed by all higher plants. As the biopolymer lignin is a major constituent of wood, it is highly abundant. Lignin biodegradation, an essential process to complete the Earth's carbon cycle, is initiated by action of several oxidoreductases excreted by white-rot fungi. The resulting degradation products may subsequently be used by other microorganisms. The non-lignolytic fungus Penicillium simplicissimum can grow on various lignin metabolites. When this ascomycete is grown on veratryl alcohol, a major lignin metabolite, production of an intracellular aryl alcohol oxidase is induced. Purification and initial characterization revealed that this enzyme is able to oxidize vanillyl alcohol into vanillin and was therefore named: vanillyl-alcohol oxidase (VAO). Furthermore, it was found that VAO is a homooctamer of about 500 kDa with each subunit containing a covalently bound 8a-( N3-histidyl)-FAD redox group. As VAO showed some interesting catalytical and structural features, a PhD-project was started in 1993 with the aim of elucidating its reaction mechanism. In the initial stage this PhD-project, it was found that VAO has a rather broad substrate specificity. However, it was unclear which substrates are of physiological relevance. In a recent study, evidence was obtained that 4-(methoxymethyl)phenol represents a physiological substrate (Chapter 2). When the fungus is grown on 4-(methoxymethyl)phenol, VAO is expressed in large amounts, while the phenolic compound is fully degraded. HPLC analysis showed that VAO catalyzes the first step in the degradation pathway of 4-(methoxymethyl)phenol (Fig. 1).Figure 1. Degradation pathway of 4-methoxymethyl)phenol in Penicillium simplicissimum.This type of reaction (breakage of an ether bond) is new for flavoprotein oxidases. Furthermore, 4-(methoxymethyl)phenol has never been described in the literature as being present in nature. Yet, it can be envisaged that this phenolic compound is formed transiently during the biodegradation of lignin, a biopolymer of phenolic moieties with many ether bonds.Concomitant with the induction of VAO a relatively high level of catalase activity was observed. A further investigation revealed that P. simplicissimum contains at least two hydroperoxidases both exhibiting catalase activities: an atypical catalase and a catalase-peroxidase (Chapter 3). Purification of both enzymes showed that the periplasmic atypical catalase contains an uncommon chlorin-type heme as cofactor. The intracellular catalase-peroxidase represents the first purified dimeric eucaryotic catalase-peroxidase. So far, similar catalase-peroxidases have only been identified in bacteria. These procaryotic hydroperoxidases show some sequence homology with cytochrome c peroxidase from yeast which is in line with their peroxidase activity. EPR experiments revealed that the catalase-peroxidase from P. simplicissimum contains a histidine as proximal heme ligand and thereby can be regarded as a peroxidase-type enzyme resembling the characterized procaryotic catalase-peroxidases.In Chapter 4, the subcellular localization of both VAO and catalase-peroxidase in P. simplicissimum was studied by immunocytochemical techniques. It was found that VAO and catalase-peroxidase are only partially compartmentalized. For both enzymes, most of the label was found in the cytosol and nuclei, while also some label was observed in the peroxisomes. The similar subcellular distribution of both oxidative enzymes suggests that catalase-peroxidase is involved in the removal of hydrogen peroxide formed by VAO. The VAO amino acid sequence revealed no clear peroxisomal targeting signal (PTS). However, the C-terminus consists of a tryptophan-lysine-leucine (WKL) sequence which resembles the well-known PTS1 which is characterized by a C-terminal serine-lysine-leucine (SKL) consensus sequence.Soon after the start of the project, it was discovered that, aside from aromatic alcohols, VAO also converts a wide range of other phenolic compounds, including aromatic amines, alkylphenols, allylphenols and aromatic methylethers (Chapter 5). Based on the substrate specificity (Fig. 2) and results from binding studies, it was suggested that VAO preferentially binds the phenolate form of the substrate. From this and the relatively high pH optimum for turnover, it was proposed that the vanillyl-alcohol oxidase catalyzed conversion of 4-allylphenols proceeds through a hydride transfer mechanism involving the formation of a p -quinone methide intermediate.Figure 2. Reactions catalyzed by VAO.In Chapter 6, the kinetic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol was studied in further detail using the stopped-flow technique. It was established that the rate-limiting step during catalysis is the reduction of the flavin cofactor by the aromatic substrate (Fig. 3). Furthermore, it was found that during this step a binary complex is formed between the reduced enzyme and a product intermediate. Spectral analysis revealed that the enzyme-bound intermediate is the p -quinone methide form of 4-(methoxymethyl)phenol. Upon reaction of this complex with molecular oxygen, the final product is formed and released in a relatively fast process. Using H218O, we could demonstrate that, upon flavin reoxidation, water attacks the electrophilic quinone methide intermediate to form the aromatic product 4-hydroxybenzaldehyde.Figure 3. Reaction mechanism for the oxidative demethylation of 4-(methoxymethyl)phenol.In Chapter 7, the enantioselectivity of VAO was investigated. VAO catalyzes the enantioselective hydroxylation of 4-ethylphenol, 4-propylphenol and 2-methoxy-4-propylphenol with an ee of 94% for the R-enantiomer. Isotope labeling experiments confirmed that the oxygen atom incorporated into the alcoholic products is derived from water. During the VAO-mediated conversion of short-chain 4-alkylphenols, 4-alkenylic phenols are produced as well. The reaction of VAO with 4-alkylphenols also results in minor amounts of phenolic ketones which is indicative for a consecutive oxidation step.Also the kinetic mechanism of VAO with 4-alkylphenols was studied (Chapter 8). For the determination of kinetic isotope effects, Ca-deuterated analogues were synthezised. Interestingly, conversion of 4-methylphenol appeared to be extremely slow, whereas 4-ethyl- and 4-propylphenol were rapidly converted. With these latter two substrates, relatively large kinetic deuterium isotope effects on the turnover rates were observed indicating that the rate of flavin reduction is rate-limiting. With all three 4-alkylphenols, the process of flavin reduction was reversible with the rate of reduction being in the same range as the rate of the reverse reaction. With 4-ethylphenol and 4-propylphenol, a transient intermediate is formed during the reductive half-reaction. From this and based on the studies with 4-(methoxymethyl)phenol, a kinetic mechanism was proposed which obeys an ordered sequential binding mechanism. With 4-ethylphenol and 4-propylphenol, the rate of flavin reduction determines the turnover rate, while with 4-methylphenol, a step involved in the reoxidation of the flavin seems to be rate limiting. The latter step might be involved in the decomposition of a flavin N5 adduct.During crystallization experiments it was found that VAO crystals are highly sensitive towards mercury and other heavy atom derivatives. Therefore, the reactivity of VAO towards mercury in solution was studied (Chapter 9). Treatment of VAO with p -mercuribenzoate showed that one cysteine residue reacts rapidly without loss of enzyme activity. Subsequently, three sulfhydryl groups react leading to enzyme inactivation and dissociation of the octamer into dimers. From this, it was proposed that subunit dissociation accounts for the observed sensitivity of VAO crystals towards mercury compounds.Recently, the crystal structure of VAO was solved (Chapter 10). The VAO structure represents the first crystal structure of a flavoenzyme with a histidyl bound FAD. The VAO monomer comprises two domains (Fig. 4).Figure 4. Crystal structure of VAO at 0.25 nm resolution.The larger domain forms a FAD-binding module while the other domain, the cap domain, covers the reactive part of the FAD cofactor. By solving the binding mode of several inhibitors, the active site of VAO could be defined. This has clarified several aspects of the catalytic mechanism of this novel flavoprotein. Three residues, Tyr108, Tyr503and Arg504, are involved in substrate activation by stabilizing the phenolate form of the substrate. This is in line with the proposed formation and stabilisation of the p -quinone methide intermediate and the substrate specificity of VAO. The structure of the enzyme 4-heptenylphenol complex revealed that the shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Furthermore, the active site cavity has a rigid architecture and is solvent-inaccessible. A major role in FAD binding is played by residues 99-110, which form the so-called 'PP loop'. This loop contributes to the binding of the adenine portion of FAD and compensates for the negative charge of the pyrophosphate moiety of the cofactor. The crystal structure also established that the C8-methyl group of the isoalloxazine ring is linked to the Ne2 atom of His422. Intriguingly, this residue is located in the cap domain.From the crystallographic data and sequence alignments, we have found that VAO belongs to a new family of structurally related flavin-dependent oxidoreductases (Chapter 11). In this study, 43 sequences were found, which show moderate homology with the VAO sequence. As sequence homology was mainly found in the C-terminal and N-terminal parts of the proteins, it could be concluded that the homology is indicative for the conservation of a novel FAD-binding domain as was found in the crystal structure of VAO (Fig. 5). This structurally related protein family includes flavin-dependent oxidoreductases isolated from (archae)bacteria, fungi, plants, animals and humans, indicating that this family is widespread. Furthermore, the sequence analysis predicts that many members of this family are covalent flavoproteins containing a histidyl bound FAD.Figure 5. Schematic drawing of the structural fold of the newly discovered flavoprotein family.Some of the VAO-mediated reactions are of relevance for the flavour and fragrance industry. For example, reactions of VAO with vanillyl alcohol, vanillylamine or creosol all result in the formation of vanillin, the major constituent of the well-known vanilla flavour. Furthermore, as shown in Chapter 7, VAO is able to enantioselectively hydroxylate phenolic compounds resulting in the production of interesting synthons for the fine-chemical industry. Because of its versatile catalytic potential and as VAO does not need external cofactors, but only uses molecular oxygen as a cheap and mild oxidant, VAO may develop as a valuable tool for the biotechnological industry. Furthermore, the recent cloning of the VAO gene and the available crystal structure will allow protein engineering to redesign the catalytic performance of VAO, which is of main interest for biotechnological applications. Therefore, like glucose oxidase andD-amino acid oxidase, VAO can be placed among an emerging group of flavoprotein oxidases, that catalyze transformations of industrial relevance.