(-ABSCISIC ACID 8'-HYDROXYLASE IS A CYTOCHROME-P450 MONOOXYGENASE())

Abscisic acid (ABA) 8'-hydroxylase catalyzes the first step in the oxi dative degradation of (+)-ABA. The development of a robust in vitro as say has now permitted detailed examination and characterization of thi s enzyme. Although several factors (buffer, cofactor, and source tissu e) were critical in developing the assay, the most important of these was the identification of a tissue displaying high amounts of in vivo enzyme activity (A.J. Cutler, T.M. Squires, M.K. Loewen, J.J. Balsevic h [1997] J Exp Bot 48: 1787-1795). (+)-ABA 8'-hydroxylase is an integr al membrane protein that is localized to the microsomal fraction in su spension-cultured maize (Zea mays) cells. (+)-ABA metabolism requires both NADPH and molecular oxygen. NADH was not an effective cofactor, a lthough there was substantial stimulation of activity (synergism) when it was included at rate-limiting NADPH concentrations. The metabolism of (+)ABA was progressively inhibited at O-2 concentrations less than 10% (v/v) and was very low (less than 5% of control) under N-2. (+)-A BA 8'-hydroxylase activity was inhibited by tetcyclacis (50% inhibitio n at 10(-6) M), cytochrome c (oxidized form), and CO. The CO inhibitio n was reversible by light from several regions of the visible spectrum , but most efficiently by blue and amber light. These data strongly su pport the contention that (+)-ABA 8'-hydroxylase is a cytochrome P450 monooxygenase.