Abstract
Cytochrome P450 monooxygenases (P450s) play crucial roles in Brassinosteroid (BR) biosynthesis from campesterol to brassinolide. Recent molecular genetic studies for BR-deficient mutants of Arabidopsis, rice, tomato and garden pea have identified several P450 genes (CYP85A, 90A, 90B, 90C, 90D, and 724B) so far. Among the BR-biosynthetic P450 genes, CYP85As and CYP90B1 have been characterized by functional expression in yeast cells and E. coli, respectively. Here, we report biochemical characterization of a P450, C-23 hydroxylase. The P450 was expressed in insect cells. The P450 activity was measured in an in vitro assay reconstituted with NADPH:cytochrome P450 reductase. The reaction products were analyzed by GC-MS. The P450 catalyzed the C-23 hydroxylation of 6-deoxocathasterone and cathasterone to 6-deoxoteasterone and teasterone, respectively. We have also determined the substrate specificity of the C-23 hydroxylase. To our knowledge, this is the first report of the biochemical characterization of the C-23 hydroxylation by the P450.
