Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols
Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols
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DOI：
10.1111/j.1365-313x.2005.02639.x
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发表时间：
2006-03-01
期刊：
影响因子：
7.2
通讯作者：
Mizutani, M
中科院分区：
文献类型：
--
作者：
Fujita, S;Ohnishi, T;Mizutani, M
拟南芥dwf 4是油菜素类固醇（BR）缺陷型突变体，并且DWF 4基因编码细胞色素P450，CYP 90 B1。我们报告的催化活性和底物特异性的CYP 90 B1。在大肠杆菌中产生重组CYP 90 B1，并在体外试验中用NADPH-细胞色素P450还原酶重构测定CYP 90 B1活性。CYP 90 B1将菜油烷醇（CN）转化为6-脱氧卡西酮，证实CYP 90 B1是类固醇C-22羟化酶。CYP 90 B1的底物特异性表明，与在C-5和C-6位没有双键的甾烷醇相比，在C-5和C-6位具有双键的甾醇是优选的底物。CYP 90 B1对菜油甾醇（CR）的催化效率（k（cat）/K-m）是CN的325倍。由于CR在植物中比CN更丰富，结果表明，在C-5 α还原之前，CR的C-22羟基化是BR生物合成途径的主要途径，这与通常接受的通过CN的途径形成对比。此外，CYP 90 B1对各种C27-29甾醇表现出C-22羟基化活性。胆固醇（C-27甾醇）是最好的底物，其次是CR（C-28甾醇），而谷甾醇（C-29甾醇）是较差的底物，这表明CYP 90 B1的底物偏好可以解释C-27/C-28/C-29甾醇和C-27/C-28/C-29 BR在植物中丰度之间的差异。
Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K-m) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5 alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C27-29 sterols. Cholesterol (C-27 sterol) is the best substrate, followed by CR (C-28 sterol), whereas sitosterol (C-29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C-27/C-28/C-29 sterols and C-27/C-28/C-29 BRs.