Brassinosteroids (BRs) are essential plant steroid hormones that regulate plant growth and development¹. The most potent BR, brassinolide, is produced by addition of many oxygen atoms to campesterol by several cytochrome P450 monooxygenases (CYPs). CYP90B1 (also known as DWF4) catalyses the 22(S)-hydroxylation of campesterol and is the first and rate-limiting enzyme at the branch point of the biosynthetic pathway from sterols to BRs². Here we show the crystal structure of Arabidopsis thaliana CYP90B1 complexed with cholesterol as a substrate. The substrate-binding conformation explains the stereoselective introduction of a hydroxy group at the 22S position, facilitating hydrogen bonding of brassinolide with the BR receptor^3,4,5. We also determined the crystal structures of CYP90B1 complexed with uniconazole^6,7 or brassinazole⁸, which inhibit BR biosynthesis. The two inhibitors are structurally similar; however, their binding conformations are unexpectedly different. The shape and volume of the active site pocket varies depending on which inhibitor or substrate is bound. These crystal structures of plant CYPs that function as membrane-anchored enzymes and exhibit structural plasticity can inform design of novel inhibitors targeting plant membrane-bound CYPs, including those involved in BR biosynthesis, which could then be used as plant growth regulators and agrochemicals.

油菜素内酯 (BRs) 是调节植物生长和发育的必需植物类固醇激素¹。最有效的 BR 芸苔素内酯是通过几种细胞色素 P450 单加氧酶 (CYP) 向菜油甾醇中添加许多氧原子而产生的。CYP90B1（也称为 DWF4）催化菜油甾醇的 22( S )-羟基化，是从甾醇到 BRs ²生物合成途径分支点的第一个限速酶。在这里，我们展示了以胆固醇为底物的拟南芥CYP90B1的晶体结构。底物结合构象解释了在 22 S处立体选择性引入羟基位置，促进油菜素内酯与 BR 受体^3,4,5的氢键结合。^{我们还确定了 CYP90B1 与烯效唑6,7}或芸苔素⁸络合的晶体结构，后者抑制 BR 生物合成。这两种抑制剂在结构上相似；然而，它们的结合构象出乎意料地不同。活性位点袋的形状和体积因结合的抑制剂或底物而异。植物 CYP 的这些晶体结构充当膜锚定酶并表现出结构可塑性，可以为设计针对植物膜结合 CYP 的新型抑制剂提供信息，包括那些参与 BR 生物合成的抑制剂，然后可以将其用作植物生长调节剂和农用化学品。