Purification and characterization of vanillyl-alcohol oxidase from Penicillium simplicissimum. A novel aromatic alcohol oxidase containing covalently bound FAD.
Vanillyl-alcohol oxidase was purified 32-fold from Penicillium simplicissimum, grown on veratryl alcohol as its sole source of carbon and energy. SDS/PAGE 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 8 alpha-(N3-histidyl)-FAD from pH-dependent fluorescence quenching (pKa = 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 vanillyl-alcohol 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.
de Jong
E
E
Department of Food Science, Agriculture University, Wageningen, The Netherlands.
van Berkel
W J
WJ
van der Zwan
R P
RP
de Bont
J A
JA
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D055592:Biophysical Phenomena; D001703:Biophysics; D002384:Catalysis; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010407:Penicillium / Q000201:enzymology*; D013057:Spectrum Analysis; D014461:Ultracentrifugation
D005182:Flavin-Adenine Dinucleotide; D000429:Alcohol Oxidoreductases; C076865:vanillyl-alcohol oxidase; D010100:Oxygen