Enzymatic assay

Enzymatic assays were performed in 200 mM K₂HPO₄-KH₂PO₄ buffer (pH 7.0), 4 mM dithiothreitol, 75 mM sodium ascorbate and 50μM substrate, unless otherwise indicated. An aliquot of 2 mM stock solution of a prenylquinone in ethanol was treated with a small grain of NaBH₄ to obtain the reduced substrate and injected into the reaction medium. The reaction was started by the addition of the recombinant enzyme (protein concentration in the stock solution was 1–1.5 mg/ml). Enzymatic assays were performed under anaerobic conditions, using oxygen trap composed of glucose oxidase, catalase and glucose [17] at 28°C for the indicated time with slow agitation.