Biosynthesis of uterotonic diterpenes from Montanoa tomentosa (zoapatle)

Montanoa tomentosa (zoapatle) is a Central American plant used in Mexico in traditional herbal medicine to ease childbirth labor and to cure certain female disorders. Recently, crude extracts of M. tomentosa have been reported to have an aphrodisiacal effect on male rats. The bioactive molecules are the uterotonic diterpenes kaurenoic acid (KA), grandiflorenic acid (GF), and monoginoic acid (MO). Roots of M. tomentosa contain all three diterpenes, whereas in leaves only kaurenoic and GF are present. However, despite the pharmacological importance of these compounds, specific information about their biosynthesis and localization in the plant is not available. In this investigation, we followed the metabolic transformation of a tritium-labeled diterpene-precursor via geranylgeranyl diphosphate into each of the three diterpenes. Inhibitors of gibberellin biosynthesis were used to elucidate the sequence of conversion of the intermediates. Our results suggest the biosynthetic conversion of KA into GF by a putative cytochrome P450-like desaturase. Partial characterization of the enzyme revealed that it requires NADPH and O2 but is inhibited by 50 microM paclobutrazol, suggesting a cytochrome P450 desaturase like enzyme (EC 1.14.14.-). Optimal reaction conditions are 32 degrees C and a pH of 7.6, respectively. Apparent kinetics parameters for KA gave a K(m,app) of 36.31 microM, and a V(max, app) of 13.6 nmol KA mg(1)protein h(-1). Based on the data presented, a putative biosynthetic pathway is proposed for the uterotonic diterpenes of M. tomentosa.