Abstract
Kaurene oxidases are P450 proteins that catalyze the conversion of ent-kaurene into kaurenoic acid, the final enzymatic product with a wide range of pharmacological properties. We describe the functional characterization of an ent-kaurene oxidase (EC 1.14.13.78) isolated from Montanoa tomentosa after heterologous expression in Saccharomyces cerevisiae, as well as the detection of the enzymatic activity in the plant itself. In the presence of NADPH and FAD, the microsomal fraction from transformed INVSc1 cells, ent-kaurene produced ent-kaurenoic acid, which was confirmed by GC-MS analyses. The kinetic parameters for ent-kaurene using 0.5 mg of microsomal protein were Km app= 80.63±1.2 μM and V max app= 31.80±1.8 μmol-1mg-1h-1. Optimal temperature and pH were 30°C and 7.6, respectively. Similar kinetic parameters were observed when leaf microsomes from M. tomentosa were assayed under the same conditions as for yeast microsomes. This result strongly suggests that ent-kaurene oxidase activity is present in leaf microsomes. The enzymatic activity was competitively inhibited by paclobutrazol, with IC50=43.9 μM, implying that MtKO is resistant to inhibition by azolic-type compounds. This study confirmed the biochemical detection of ent-kaurene oxidase activity in the plant, and the heterologous functionality of a cDNA with an ent-kaurene oxidase identity from M. tomentosa (zoapatle).
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