Purification and characterization of an enzyme that catalyzes ring cleavage of aspergillic acid, from Trichoderma koningii ATCC 76666

The aspergillic acid degrading enzyme (ADE) that catalyzes the cleavage of the pyrazine ring in aspergillic acid (AA, 1-hydroxy-3-isobutyl-6-sec-butyl-2-pyrazinone) was purified to electrophoretic homogeneity from extracts of Trichoderma koningii ATCC 76666. ADE was a homodimeric protein with a molecular mass of 112 kDa, contained 1 mol of FAD per mol of subunit, and required NAD(P)H and molecular oxygen for its activity. ADE had an isoelectric point of around 5.3, and an optimum pH of 7.0-8.0. p-Chloromercuribenzoate and HgCl2 completely inhibited ADE activity, while metal chelating reagents, α, α'-dipyridyl and o-phenanthroline, were not inhibitors. The substrate specificity among AA-related compounds was that hydroxyaspergillic acid was a poor substrate (16% of the activity for AA) and deoxyaspergillic acid did not serve as a substrate.