Partial purification and characterization of a dehydratase associated with the pentaketide melanogenesis pathway of Phaeococcomyces sp. and other fungi

Abstract

Polyacrylamide gel electrophoresis was used to identify an enzyme that catalyzed the dehydration of scytalone to 1,3,8-trihydroxynaphthalene, the next sequential component of the pentaketide melanin pathway. The dehydratase, extracted from the pentaketide melanin-producing black yeast Phaeococcomyces sp., was partially purified by gel filtration and preparative electrophoresis. Enzyme activity was indicated by the formation of a single orange band on polyacrylamide gels after scytalone was added as substrate. The enzyme has a molecular mass of ca. 60,000–65,000 and an isoelectric point of 5.2. The pH optimum for activity with scytalone under aerobic conditions was 7.5. Anaerobic reactions resulted in the accumulation of 1,3,8-trihydroxynaphthalene, and aerobic reactions produced 2-hydroxyjuglone, which is an orange-colored autooxidation product of 1,3,8-trihydroxynaphthalene. Reduced nucleotides were not required for the dehydratase reaction. When the pentaketide pathway intermediate vermelone was used as a substrate it was converted to 1,8-dihydroxynaphthalene and an unknown hydroxynaphthalene. Extracts of the plant pathogenic pentaketide melanin-producing fungi Verticillium dahliae, Verticillium albo-atrum, Alternaria solani, and Cladosporium cucumerinum were also shown to have the dehydratase activity on polyacrylamide electrophoresis gels. The dehydratase activity did not occur with extracts of yeasts that do not form pentaketide melanins or with extracts from V. dahliae and V. albo-atrum that had not yet formed melanin.