Cytochrome P450 CYP710A encodes the sterol C-22 desaturase in Arabidopsis and tomato.
Delta22-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 beta-sitosterol to produce stigmasterol (CYP710A1, K(m) = 1.0 microM and kinetic constant [k(cat)] = 0.53 min(-1); CYP710A11, K(m) = 3.7 microM 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 beta-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:beta-glucuronidase transgenic approaches indicated strict tissue/organ-specific regulation for each CYP710A gene, implicating differential tissue distributions of the Delta(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.
Morikawa
Tomomi
T
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
Mizutani
Masaharu
M
Aoki
Nozomu
N
Watanabe
Bunta
B
Saga
Hirohisa
H
Saito
Shigeki
S
Oikawa
Akira
A
Suzuki
Hideyuki
H
Sakurai
Nozomu
N
Shibata
Daisuke
D
Wadano
Akira
A
Sakata
Kanzo
K
Ohta
Daisaku
D
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics; D017124:Conserved Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012855:Sitosterols / Q000378:metabolism; D013261:Sterols / Q000096:biosynthesis; D013265:Stigmasterol / Q000378:metabolism
D029681:Arabidopsis Proteins; D010940:Plant Proteins; D012855:Sitosterols; D013261:Sterols; C025473:gamma-sitosterol; D003577:Cytochrome P-450 Enzyme System; D013265:Stigmasterol; D010088:Oxidoreductases