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. 2012 Sep 7;287(37):31551-60.
doi: 10.1074/jbc.M112.392720. Epub 2012 Jul 20.

CYP90A1/CPD, a brassinosteroid biosynthetic cytochrome P450 of Arabidopsis, catalyzes C-3 oxidation

Affiliations

CYP90A1/CPD, a brassinosteroid biosynthetic cytochrome P450 of Arabidopsis, catalyzes C-3 oxidation

Toshiyuki Ohnishi et al. J Biol Chem. .

Abstract

Brassinosteroids (BRs) are steroidal phytohormones that regulate plant growth and development. Whereas in Arabidopsis the network-like routes of BR biosynthesis have been elucidated in considerable detail, the roles of some of the biosynthetic enzymes and their participation in the different subpathways remained to be clarified. We investigated the function of the cytochrome P450 monooxygenase CYP90A1/CPD, which earlier had been proposed to act as a BR C-23 hydroxylase. Our GC-MS and genetic analyses demonstrated that the cpd mutation arrests BR synthesis upstream of the DET2-mediated 5α reduction step and that overexpression of the C-23 hydroxylase CYP90C1 does not alleviate BR deficiency in the cpd mutant. In line with these results, we found that CYP90A1/CPD heterologously expressed in a baculovirus-insect cell system catalyzes C-3 oxidation of the early BR intermediates (22S)-22-hydroxycampesterol and (22R,23R)-22,23-dihydroxycampesterol, as well as of 6-deoxocathasterone and 6-deoxoteasterone. Enzyme kinetic data of CYP90A1/CPD and DET2, together with those of the earlier studied CYP90B1, CYP90C1, and CYP90D1, suggest that BR biosynthesis proceeds mainly via the campestanol-independent pathway.

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Figures

FIGURE 1.
FIGURE 1.
BR biosynthetic pathways, including the late C-6, early C-22, and C-23 oxidation routes. The subpathway indicated by dotted lines has not been verified in planta. BL, brassinolide; CN, campestanol; CR, campesterol; CS, castasterone; CT, cathasterone; TE, teasterone; TY, typhasterol; 22-OHCR, (22S)-22-hydroxycampesterol; 22-OH-3-one, (22S)-22-hydroxy-5α-campestan-3-one; 22-OH-4-en-3-one, (22S)-22-hydroxycampest-4-en-3-one; 22,23-diOHCR, (22R,23R)-22,23-dihydroxycampesterol; 22,23-diOH-4-en-3-one, (22R,23R)-22,23-dihydroxycampest-4-en-3-one; 3-epi-6-deoxoCT, 3-epi-6-deoxocathasterone; 3-one, 5α-campestan-3-one; 4-en-3-one, campest-4-en-3-one; 6-deoxoCS, 6-deoxocastasterone; 6-deoxoCT, 6-deoxocathasterone; 6-deoxoTE, 6-deoxoteasterone; 6-deoxoTY, 6-deoxotyphasterol; 6-deoxo3DT, 3-dehydro-6-deoxoteasterone.
FIGURE 2.
FIGURE 2.
Phenotypic consequences of CYP90A1/CPD deficiency are not affected by CYP90C1 gene activity or the det2 mutation. A, CPD promoter-driven overexpression of CYP90C1 in cpd mutant background. One-month-old aseptically grown cpd mutant and CPD:CYP90C1-expressing transgenic plant are shown. Bar, 10 mm. B, semiquantitative RT-PCR detection of the CPD, CYP90C1, and the standard UBQ10 transcripts in wild type Arabidopsis (Col-0), the cpd mutant, and the CPD:CYP90C1 transgenic line with cpd background. C, phenotypes of the cpd, det2, and cpd/det2 mutants. One-month-old soil-grown plantlets. Bar, 10 mm.
FIGURE 3.
FIGURE 3.
GC-MS analysis of BR metabolites produced by heterologously expressed CYP90A1/CPD. Selected ion chromatograms of the reaction products were obtained with 6-deoxoCT (fragment ion for detection at m/z 97 and 187) (A), 22-OHCR (fragment ion for detection at m/z 97 and 187) (B), 6-deoxoTE (fragment ion for detection at m/z 155 and 231) (C), and 22,23-OHCR (fragment ion for detection at m/z 124 and 229) (D) as substrates. C-3 oxidation of BR biosynthetic intermediates by CYP90A1/CPD (E).
FIGURE 4.
FIGURE 4.
Relative activities of CYP90A1/CPD with 6-deoxoCT, 22-OHCR, 6-deoxoTE, and 22,23-diOHCR substrates. CYP90A1/CPD assay mixtures were supplemented with BRs (to 20 μm), incubated at 30 °C for 20 min, and the reaction products were then analyzed by GC-MS. The activity obtained with 22-OHCR substrate (100%) corresponds to 2.27 units (defined as pmol of product/min/pmol of P450). Values were determined in four replicate measurements; error bars indicate standard deviation.
FIGURE 5.
FIGURE 5.
CN-independent and CN-dependent routes of BR biosynthesis. The CN-independent pathway, via CR → 22-OHCR → 22-OH-4-en-3-one → 22-OH-3-one → 3-epi-6-deoxoCT → 6-deoxoTY → 6-deoxoCS → CS → BL, is two reactions shorter than the CN-dependent route, due to shortcutting the 3-one → CN and 6-deoxoTE → 6-deoxo3DT reactions.

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