Purification and characterization of dimethylallyl pyrophosphate:aspulvinone dimethylallyltransferase from Aspergillus terreus

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Abstract
Dimethylallyl pyrophosphate:aspulvinone dimethylallyltransferase, the prenylation enzyme for the biosynthesis of aspulvinone pigments, was purified from mycelia of A. terreus. The transferase catalyzed the transfer of the dimethylallyl moiety from dimethylallyl pyrophosphate to either of the 2 aromatic rings of aspulvinone E to give the mono- and dipyrenylated derivatives which were identified with the metabolites aspulvinone I and aspulvinone H, respectively. Aspulvinnone G, another fundamental metabolite of this series, also acted as substrate to afford the corresponding diprenylated derivative, which is assumed to be a precursor for aspulvinone C, D, and F. The MW of the enzyme was estimated to be 240,000-270,000 by gel filtration. Since the subunit MW determined by NaDodSO4-polyacrylamide disc gel electrophoresis was 45,000, the native enzyme appears to be a hexomeric protein composed of identical MW subunits. The apparent Km values for aspulvinone E, aspulvinone G and dimethylallyl pyrophosphate were 13.7, 7.7 and 40.0 .mu.M, respectively. The enzyme shows the maximum activity at pH 7.0, and no metal ion is necessary for the activation. Sulfhydryl blocking agents or mercaptoethanol has no effect. Bromophenol blue binds specifically, to the transferase and strongly inhibits the enzyme activity.