Degradation of veratryl alcohol by Penicillium simplicissimum

Jong, Ed de; Beuling, E.E.; Zwan, R.P. van der; Bont, J.A.M. de

doi:10.1007/bf00170072

Cited by 31 publications

(25 citation statements)

References 23 publications

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“…simplicissimum was routinely grown fed batch in a flatbottomed round flask with veratryl alcohol as sole carbon and energy source (de Jong et al, 1990). Under these conditions, an intracellular vanillyl-alcohol-oxidizing activity was induced.…”

Section: Enzyme Purificationmentioning

confidence: 99%

“…CBS 170.90 was maintained at 4°C on malt-agar slants. The fungus was cultivated on veratryl alcohol in a fed-batch fermentor as described before (de Jong et al, 1990). Under these conditions, vanillylalcohol oxidase was induced, oxidizing vanillyl alcohol at 75 f 2 0 nmol .…”

Section: Enzyme Purificationmentioning

confidence: 99%

“…Veratryl alcohol plays a crucial role in lignin degradation by the white-rot fungus Phanerochaete chrysosporium (Buswell, 1992). P. simplicissimum degrades veratryl alcohol via NAD(P)+-dependent dehydrogenase(s) and veratric-acid 0-demethylase(s) (de Jong et al, 1990). Recently it was found that this organism, when grown on veratryl alcohol, induces an intracellular H202-generating oxidase not acting on veratryl alcohol but rather on vanillyl alcohol.…”

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confidence: 99%

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Purification and characterization of vanillyl‐alcohol oxidase from Penicillium simplicissimum

Jong¹,

Berkel

Zwan

et al. 1992

European Journal of Biochemistry

136

109

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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

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Section: Enzyme Purificationmentioning

confidence: 99%

Section: Enzyme Purificationmentioning

confidence: 99%

mentioning

confidence: 99%

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Purification and characterization of vanillyl‐alcohol oxidase from Penicillium simplicissimum

Jong¹,

Berkel

Zwan

et al. 1992

European Journal of Biochemistry

136

109

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“…Vanillyl-alcohol oxidase (VAO) was discovered in 1990 during studies on the biodegradation of aromatic compounds by the fungus P. simplicissimum [4]. This ascomycete, isolated from paper mill wastewater, was selected for its ability to use veratryl alcohol, a key compound in lignin biodegradation as the sole source of carbon and energy.…”

Section: Introductionmentioning

confidence: 99%

“…From proteolytic fragmentation of the polypeptide chain and fluorescence analysis, the flavin cofactor was identified as 8a-(N 3 )-histidyl) -FAD. Although the expression of VAO is induced by veratryl alcohol, the enzyme is not involved in the catabolism of veratryl alcohol in P. simplicissimum [4]. This raised the question about the physiological significance of VAO.…”

Section: Introductionmentioning

confidence: 99%

Structure, function and redesign of vanillyl-alcohol oxidase

Heuvel

Fraaije

Mattevi

et al. 2002

International Congress Series

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Vanillyl-alcohol oxidase (VAO) from Penicillium simplicissimum is an inducible flavoprotein involved in the biodegradation of lignin-derived aromatic compounds. The enzyme is the prototype of a newly recognized family of structurally related oxidoreductases, whose members share a conserved FAD-binding domain. The flavin cofactor in VAO is covalently linked to His422 of the cap domain. This covalent interaction increases the redox potential of the flavin, facilitating the oxidation of a wide range of phenolic substrates. Covalent flavinylation is an autocatalytic process in which His61 activates His422. Vanillyl-alcohol oxidase (VAO) catalysis involves the formation of a p-quinone methide product intermediate. With 4-ethylphenol as a substrate, this intermediate is stereospecifically attacked by water, yielding the R-enantiomer of 1-(4V -hydroxyphenyl)ethanol in high enantiomeric excess. Studies from site-directed mutants revealed that Asp170 is critically involved in substrate hydroxylation. This prompted us to create a double mutation (D170S/T457E) with the idea to relocate the putative active site base to the opposite face of the substrate-binding pocket. In this way, and for the first time, the stereospecificity of a redox enzyme was inversed by rational redesign. D

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Crystallization and preliminary x-ray analysis of the flavoenzyme vanillyl-alcohol oxidase fromPenicillium Simplicissimum

et al. 1997

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Vanillyl-alcohol oxidase catalyses the oxidation of several 4-hydroxybenzyl alcohols by using 8-a-(N 3 -histidyl)-FAD as a covalently bound prosthetic group. Crystals of vanillyl-alcohol oxidase from Penicillium simplicissimum have been grown by using the vapor diffusion technique. The space group was found to be I4, with cell dimensions a 5 b 5 140.5 Å, c 5 132.9 Å. Diffraction data have been recorded to 3.2 Å resolution by using a laboratory source and to 2.5 Å resolution on flash freezing the crystal at the ELETTRA Synchrotron X-ray diffraction beam line. Proteins 27:

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Degradation of veratryl alcohol by Penicillium simplicissimum

Cited by 31 publications

References 23 publications

Purification and characterization of vanillyl‐alcohol oxidase from Penicillium simplicissimum

Purification and characterization of vanillyl‐alcohol oxidase from Penicillium simplicissimum

Structure, function and redesign of vanillyl-alcohol oxidase

Crystallization and preliminary x-ray analysis of the flavoenzyme vanillyl-alcohol oxidase fromPenicillium Simplicissimum

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