Purification and characterization of vanillyl‐alcohol oxidase from Penicillium simplicissimum

Abstract

Vanillyl-alcohol oxidase was purified 32-fold from Penicillium simplicissimum, grown on veratryl alcohol as its sole source of carbon and energy. SDSjPAGE of the purified enzyme reveals a single fluorescent band of 65 kDa. Gel filtration and sedimentation-velocity experiments indicate that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8cr-(N3-histidyl)-FAD from pH-dependent fluorescence quenching (pK, = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride.The enzyme shows a narrow substrate specificity, only vanillyl alcohol and 4-hydroxybenzyl alcohol are substrates for the enzyme. Cinnamyl alcohol is a strong competitive inhibitor of vanillylalcohol oxidation.The visible absorption spectrum of the oxidized enzyme shows maxima at 354 nm and 439 nm, and shoulders at 370, 417 and 461 nm. Under anaerobic conditions, the enzyme is easily reduced by vanillyl alcohol to the two-electron reduced form. Upon mixing with air, rapid reoxidation of the flavin occurs. Both with dithionite reduction and photoreduction in the presence of EDTA and 5-deazaflavin the red semiquinone flavin radical is transiently stabilized. Opposite to most flavoprotein oxidases, vanillyl-alcohol oxidase does not form a flavin N5-sulfite adduct. Photoreduction of the enzyme in the presence of the competitive inhibitor cinnamyl alcohol gives rise to a complete, irreversible bleaching of the flavin spectrum.Fungal aryl-alcohol oxidases (aromatic-alcohol oxidase) described to date include enzymes from Polystictus versicolor (Farmer et al., 1960), Pleurotus sajor-caju (Bourbonnais and Paice, 1988), Pleurotus eryngii (Guillkn et al., 1990), Pleurotus ostreatus (Sannia et al., 1991), Bjerkandera adusta (Muheim et al., 1990) and from Fusariurn sp. (Iwahara et al., 1980). All the extracellular aryl-alcohol oxidases excreted by the abovementioned white-rot fungi have a rather broad substrate specificity. It is thought that these aryl-alcohol oxidases play a role in HzOz production, necessary in lignin biodegradation. The aryl-alcohol oxidases of B. adusta and P. ostreatus are both flavoproteins, containing non-covalently bound FAD as prosthetic group.The fungus Penicillium simplicissimum CBS 170.90 is not ligninolytic but can use a range of aromatic compounds, including veratryl and vanillyl alcohol, as sole source

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

“…The concentration of VAO was determined spectrophotometrically using a molar absorption coefficient E439 = 12.5 mM _1 cm -1 for protein-bound FAD [1]. VAO substrates and competitive inhibitors have been described elsewhere [2].…”

“…The enzyme is a homooctamer of 0.5 MDa with each subunit containing Soc-iTV^-histidyrj-FAD as a covalently bound prosthetic group [1]. Based on the production of 4-hydroxycinnamyl alcohols from 4-allylphenols we have proposed that the reaction mechanism of VAO involves the formation of a />-quinone methide product intermediate [2].…”

“…Vanillyl-alcohol oxidase (VAO) from Penicillium simplicissimum catalyzes the oxidation of vanillyl-alcohol to vanillin with the simultaneous reduction of molecular oxygen to hydrogen peroxide [1]. The enzyme is a homooctamer of 0.5 MDa with each subunit containing Soc-iTV^-histidyrj-FAD as a covalently bound prosthetic group [1].…”

Inversion of stereospecificity of vanillyl-alcohol oxidase

“…The purified VAO mutants T457E, D170A͞T457E, and D170S͞T457E were bright yellow, and their near UV absorption properties were virtually identical to wildtype enzyme (29). Fluorescence analysis of unstained SDS gels showed that the mutant enzymes were as fluorescent as wildtype VAO.…”

“…Fluorescence analysis of unstained SDS gels showed that the mutant enzymes were as fluorescent as wildtype VAO. This fluorescence behavior, and the fact that the mutants did not release any flavin upon trichloroacetic acid treatment (29), indicated that all flavin was covalently bound. Moreover, analytical gel filtration revealed that the mutants were mainly octameric, with only a small fraction in the dimeric form.…”