Laccase--and not tyrosinase--is the enzyme responsible for quinone methide production from 2,6-dimethoxy-4-allyl phenol.
Tyrosinase, which is known to possess both monophenol monooxygenase activity (EC 1.14.18.1, tyrosine, 3,4-dihydroxyphenylalanine:oxygen oxidoreductase) and o-diphenoloxidase activity (EC 1.10.3.1, o-diphenol:oxygen oxidoreductase), has been shown to exhibit other related activities. Recently, a new reaction, viz., oxidative conversion of 2,6-dimethoxyallyl phenol to its quinone methide, catalyzed by commercial preparations of mushroom tyrosinase was reported (E. S. Krol, and J. L. Bolton, 1997, Chem. Biol. Interact. 104, 11-27). Since the reaction involves an unusual 1,6-oxidation rather than the conventional 1,4-oxidation, we reexamined this reaction more carefully. The o-diphenoloxidase activity and the dimethoxyallyl phenol oxidase activity of mushroom tyrosinase preparations exhibited different mobilities on size-exclusion chromatography on a Sephacryl S-200 column. A similar behavior was also witnessed on preparative isoelectric focusing in a rotofor cell. Different preparations of mushroom tyrosinase possessed varying ratios of these two activities, further confirming that they are due to two different enzymes. Native polyacrylamide gel electrophoresis followed by activity staining of the gel revealed different mobilities for these two activities. The protein band exhibiting dimethoxyallyl phenol oxidase activity could also be stained by syringaldazine, a well-known substrate for laccase (EC 1.10.3.2, p-diphenol:oxygen oxidoreductase). Two insect phenoloxidases, which are known for their wide substrate specificity, failed to oxidize dimethoxyallyl phenol to any detectable extent, thereby confirming that typical o-diphenoloxidases lack the ability to oxidize dimethoxyallyl phenol. On the other hand, laccase, which is known to convert syringaldazine to its quinone methide derivative, readily produced the quinone methide from dimethoxyallyl phenol. It is therefore concluded that laccase, which is present as a contaminant in the commercial preparations of mushroom tyrosinase--and not tyrosinase (o-diphenoloxidase)--is the enzyme responsible for catalyzing the new conversion of dimethoxyallyl phenol to its corresponding quinone methide.
Sugumaran
M
M
Department of Biology, University of Massachusetts at Boston 02125, USA. sugumaran@umbsky.cc.umb.edu
Bolton
J L
JL
D001487:Basidiomycota / Q000201:enzymology; D002850:Chromatography, Gel; D004340:Drug Contamination; D005054:Eugenol / Q000031:analogs & derivatives* / Q000378:metabolism; D045563:Indolequinones; D007211:Indoles / Q000378:metabolism*; D007525:Isoelectric Focusing; D042845:Laccase; D008956:Models, Chemical; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism*; D013379:Substrate Specificity
D045563:Indolequinones; D007211:Indoles; D011809:Quinones; C068040:quinone methide; D005054:Eugenol; C112468:4-allyl-2,6-dimethoxyphenol; D010088:Oxidoreductases; D042845:Laccase; D014442:Monophenol Monooxygenase