Purification and properties of dihydrogeodin oxidase from Aspergillus terreus.

The last step of (+)-geodin biosynthesis is a phenol oxidative coupling, which is one of the most important reactions in biosynthesis of natural products. The enzyme named dihydrogeodin oxidase catalyzes the regio- and stereospecific phenol oxidative coupling reaction to form (+)-geodin from dihydrogeodin. The enzyme was purified from the cell-free extract of Aspergillus terreus, a (+)-geodin producer, by ammonium sulfate fractionation, acid treatment, and column chromatographies on DEAE-cellulose, Hydroxyapatite, chromatofocusing, and Toyopearl HW-55S. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 153,000 by gel filtration on a Toyopearl HW-55S column and 76,000 by SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer. The purified enzyme showed an intense blue color and had absorption maxima at 280 and 600 nm, which suggested it to be a blue copper protein. The copper content was found to be 8 atoms per subunit by atomic absorption analysis and no significant amount of other metals was detected by ICP emission spectrometry. The electron paramagnetic resonance spectrum showed the presence of type 1 and type 2 copper atoms in the enzyme molecule. Sodium azide and ethylxanthate inhibited the enzyme activity, but potassium cyanide and diethyldithiocarbamate, both known as potent copper enzyme inhibitors, were not inhibitory.