Structural insights into a key step of brassinosteroid biosynthesis and its inhibition

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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 receptor3,4,5. We also determined the crystal structures of CYP90B1 complexed with uniconazole6,7 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.

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Fig. 1: Crystal structure of cholesterol-bound CYP90B1 and docking simulation of campesterol.
Fig. 2: Enzymatic activity of CYP90B1.
Fig. 3: Crystal structure of uniconazole-bound CYP90B1.
Fig. 4: Crystal structure of brassinazole-bound CYP90B1.

Data availability

Atomic coordinates and crystallographic structure factors have been deposited in the Protein Data Bank under accession codes 6A15 (cholesterol-bound form), 6A16 (uniconazole-bound form), 6A17 (brassinazole-bound form) and 6A18 (1,6-hexanediol-bound form). All other data are available from the corresponding author upon reasonable request.


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We thank K. Okamoto for constructing various expression plasmids for CYP90B1 and for purification of this enzyme. We also thank the beamline staff of BL26B1, BL41XU and BL32XU at SPring-8 (Hyogo, Japan) for assistance with the experiments. This study was supported by JSPS KAKENHI grant number JP17H05444, JP17K05933 and JP18H03945. This study is partially supported by the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science (MEXT) and Japan Agency for Medical Research and Development (AMED) under grant number JP18am0101070.

Author information

K.F., T.H. and M.K. performed enzyme purification and crystallography. K.F., T.H., M.M. and S.N. wrote the manuscript. B.W., H.J.L. and M.M. determined enzyme activities. All authors discussed the data.

Correspondence to Shingo Nagano.

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The authors declare no competing interests.

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Peer review information: Nature Plants thanks Michael Hothorn and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–17 and Supplementary Tables 1–2.

Reporting Summary

Supplementary Video 1

Video showing cholesterol-binding mode.

Supplementary Video 2

Video showing uniconazole-binding mode.

Supplementary Video 3

Video showing brassinazole-binding mode

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Fujiyama, K., Hino, T., Kanadani, M. et al. Structural insights into a key step of brassinosteroid biosynthesis and its inhibition. Nat. Plants 5, 589–594 (2019) doi:10.1038/s41477-019-0436-6

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