Asp-170 Is Crucial for the Redox Properties of Vanillyl-alcohol Oxidase

Abstract

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. Vanillyl-alcohol oxidase (VAO)1 (EC 1.1.3.38) from Penicillium simplicissimum is a homooctameric covalent flavoenzyme involved in the biodegradation of lignin-derived aromatic compounds (1). The enzyme is the prototype of a novel family of structurally related oxidoreductases sharing a conserved FADbinding domain (2). VAO oxidizes its physiological substrate 4-(methoxymethyl)phenol to 4-hydroxybenzaldehyde with the concomitant reduction of molecular oxygen to hydrogen peroxide (3, 4). The enzymatic reaction is initiated by the transfer of a hydride equivalent from the C␣-atom of the substrate to flavin N5. The resulting binary complex between the reduced enzyme and the p-quinone methide intermediate then reacts with molecular oxygen, reoxidizing the FAD. The p-quinone methide product subsequently reacts with water in the enzyme active site to generate the final products 4-hydroxybenzaldehyde and methanol (4) (Fig. 1). A similar reaction mechanism has been proposed for the structurally related flavocytochrome p-cresol methylhydroxylase (5, 6).Recently, we determined the three-dimensional structures of native VAO and several enzyme-ligand complexes (7). Each VAO 64-kDa monomer consists of two domains: the cap domain covers the active site, whereas the larger domain creates a binding site for the ADP-ribityl part of the FAD cofactor. The C8␣-atom of the isoalloxazine ring of the FAD is covalently linked to the N⑀3-ato...

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

“…Error-prone PCR Mutagenesis-Plasmid pBC11 contains the vao gene with a silent mutation at position 882, introducing a SalI restriction site (33). For random mutagenesis, the vao gene in pBC11 was amplified using manganese-based error-prone PCR mutagenesis (34,35).…”

“…The seven clones with the highest activity for creosol were sequenced. Wild-type VAO and its mutants were overexpressed and purified according to a previously established procedure (33,36). The enzyme purity was checked by SDS-PAGE and by size-exclusion chromatography.…”

Structure of a flavin-binding plant photoreceptor domain: Insights into light-mediated signal transduction

“…A hydrogen bond to N5 is a recurrent feature in most f lavoenzymes (42) but is absent in vannilyl-alcohol oxidase (43) and glycolate oxidase (44). Although both of these f lavoproteins exhibit a higher-than-average redox potential (45,46), the consequences of the absence of a hydrogen bond at this position are not clear.…”

Inversion of stereospecificity of vanillyl-alcohol oxidase

“…The side chain of this acidic residue is only 3.6 Å from flavin N5 and the reactive methylene group of the phenolic substrate (8). From studies on Asp-170 variants, we recently showed that Asp-170 is important for maintaining the high redox potential of the enzyme and thus its reactivity (11). Furthermore, these studies revealed that Asp-170 assists in covalent flavinylation, possibly by donating a proton to flavin N5 (11).…”

“…From studies on Asp-170 variants, we recently showed that Asp-170 is important for maintaining the high redox potential of the enzyme and thus its reactivity (11). Furthermore, these studies revealed that Asp-170 assists in covalent flavinylation, possibly by donating a proton to flavin N5 (11). It has been suggested that, in the related flavocytochrome PCMH, Glu-380 fulfills a similar role during the flavinylation process (4,11,12).…”