C-23 Hydroxylation byArabidopsisCYP90C1 and CYP90D1 Reveals a Novel Shortcut in Brassinosteroid Biosynthesis

Abstract

Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5a-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5a-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.

CYP90A1/CPD, a Brassinosteroid Biosynthetic Cytochrome P450 of Arabidopsis, Catalyzes C-3 Oxidation

“…Recently, however, we obtained biochemical and genetic evidence that in Arabidopsis this hydroxylation reaction is catalyzed by the redundantly functioning CYP90C1 and CYP90D1 enzymes (13). As cyp90c1/cyp90d1 double null mutants, carrying a fully functional CYP90A1/CPD gene, show severe BRdeficient dwarf phenotypes (13) similar to that of cpd, this was a clear indication that the role of CYP90A1/CPD should be different from those of CYP90C1 and CYP90D1 and other than C-23 hydroxylation.…”

“…For instance, the cpd mutant, lacking CYP90A1/ CPD, was rescued by the 22,23-dihydroxylated BRs teasterone, 3-dehydroteasterone, typhasterol, castasterone (CS), and BL but not by the 22-hydroxylated cathasterone (CT), which led to the suggestion that the mutation impairs C-23 hydroxylation (9). Recently, however, we obtained biochemical and genetic evidence that in Arabidopsis this hydroxylation reaction is catalyzed by the redundantly functioning CYP90C1 and CYP90D1 enzymes (13). As cyp90c1/cyp90d1 double null mutants, carrying a fully functional CYP90A1/CPD gene, show severe BRdeficient dwarf phenotypes (13) similar to that of cpd, this was a clear indication that the role of CYP90A1/CPD should be different from those of CYP90C1 and CYP90D1 and other than C-23 hydroxylation.…”

“…1) where broad substrate specificities of the participating enzymes allow metabolite flow via multiple pathways (13)(14)(15). Additionally, most BR biosynthetic genes are up-regulated in BR-deficient mutants (16,17), thereby increasing the role of subsidiary reaction routes.…”

“…Additionally, most BR biosynthetic genes are up-regulated in BR-deficient mutants (16,17), thereby increasing the role of subsidiary reaction routes. Because of these intricacies, the use of indirect methods for the functional characterization of BR biosynthetic genes can be difficult and misleading, as has been the case with the cyp90c1 and cyp90d1 mutants (12,13).…”

“…gest that CYP90A1/CPD-driven BR synthesis proceeds mainly via the early C-22 hydroxylation pathway, facilitating the use of the campestanol (CN)-independent route, which allows direct conversion of early 22-hydroxylated intermediates to 6-deoxo3DT and 6-deoxotyphasterol (6-deoxoTY) via C-23 hydroxylation (13).…”