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Cytochrome P450 CYP710A Encodes the Sterol C-22 Desaturase in Arabidopsis and Tomato^[W],[OA]

^a Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
^b Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
^c Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan

² To whom correspondence should be addressed. E-mail ohtad{at}bioinfo.osakafu-u.ac.jp; fax 81-72-254-9409.

22-Unsaturated sterols, containing a double bond at the C-22 position in the side chain, occur specifically in fungi and plants. Here, we describe the identification and characterization of cytochrome P450s belonging to the CYP710A family as the plant C-22 desaturase. Recombinant proteins of CYP710A1 and CYP710A2 from Arabidopsis thaliana and CYP710A11 from tomato (Lycopersicon esculentum) were expressed using a baculovirus/insect system. The Arabidopsis CYP710A1 and tomato CYP710A11 proteins exhibited C-22 desaturase activity with ß-sitosterol to produce stigmasterol (CYP710A1, K_m = 1.0 µM and kinetic constant [k_cat] = 0.53 min^–1; CYP710A11, K_m = 3.7 µM and k_cat = 10 min^–1). In Arabidopsis transgenic lines with CYP710A1 and CYP710A11 overexpression, stigmasterol levels increased by 6- to 32-fold. Arabidopsis CYP710A2 was able to produce brassicasterol and stigmasterol from 24-epi-campesterol and ß-sitosterol, respectively. Sterol profiling analyses for CYP710A2 overexpression and a T-DNA insertion event into CYP710A2 clearly demonstrated in planta that CYP710A2 was responsible for both brassicasterol and stigmasterol production. Semiquantitative PCR analyses and promoter:ß-glucuronidase transgenic approaches indicated strict tissue/organ-specific regulation for each CYP710A gene, implicating differential tissue distributions of the ²²-unsaturated sterols in Arabidopsis. Our results support the possibility that the CYP710 family may encode P450s of sterol C-22 desaturases in different organisms.

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