Abstract
Brassinosteroids (BRs) are thought to be biosynthesized from campesterol via campestanol in plants. Recently, we have characterized that Arabidopsis CYP90C1 and CYP90D1 are redundant C-23 hydroxylases. Analyses of their substrate specificities revealed a novel shortcut in BR biosynthesis, which allows direct conversion of early 22-hydroxylated intermediates to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol via C-23 hydroxylation. Here, we report biochemical characterization of this novel shortcut, focusing on 5α-reductase (DET2) and C-3 oxidase. These recombinant enzymes were heterogenously expressed, and their activities were measured in vitro. DET2 catalyzed 5α-reduction of not only (24R)-ergost-4-en-3-one (4-en-3-one) but also 22-hydroxy-4-en-3-one and 22,23-dihydroxy-4-en-3-one. The substrate specificity of C-3 oxidase showed that (22S)-22-hydroxycampesterol is a better substrate than (22R,23R)-22,23-hydroxycampesterol, whereas campesterol is not metabolized. Thus, the shortcut is likely a main route of BR biosynthetic pathway.
