CYP710A genes encoding sterol C22-desaturase in Physcomitrella patens as molecular evidence for the evolutionary conservation of a sterol biosynthetic pathway in plants
CYP710A genes encoding sterol C22-desaturase in Physcomitrella patens as molecular evidence for the evolutionary conservation of a sterol biosynthetic pathway in plants
复制标题
DOI：
10.1007/s00425-009-0916-4
复制
发表时间：
2009-05-01
期刊：
影响因子：
4.3
通讯作者：
Ohta, Daisaku
中科院分区：
文献类型：
--
作者：
Morikawa, Tomomi;Saga, Hirohisa;Ohta, Daisaku
我们的特点是细胞色素P450，CYP 710 A13和CYP 710 A14，作为甾醇C22-去饱和酶的苔藓小立碗藓。GC-MS分析表明，P. patens积累的豆甾醇为主要甾醇（占总甾醇的56-60%），谷甾醇较少（8-12%）;这种甾醇谱与高等植物积累的豆甾醇为次要组分形成对比。利用杆状病毒/昆虫细胞系统制备的重组CYP 710 A13和CYP 710 A14蛋白表现出与β-谷甾醇产生豆甾醇的C22-去饱和酶活性，而菜油甾醇和24-表-菜油甾醇不被接受为底物。CYP 710 A13（1.0 +/- A 0.043 μ M）和CYP 710 A14（2.1 +/- A 0.17 μ M）的β-谷甾醇的K（m）值与报道的较高植物CYP 710 A蛋白的K（m）值相当。在过表达CYP 710 A14的拟南芥T87细胞中，豆甾醇含量达到了比T87细胞基础水平高20- 72倍的水平，证实了这种P450酶的C22-去饱和酶活性。最终产物的出现以及参与途径最后一步的酶的存在证实了在P. patens中存在完整的甾醇生物合成途径，为陆生植物的进化过程中甾醇生物合成途径的保护提供了证据。
We have characterized cytochromes P450, CYP710A13, and CYP710A14, as the sterol C22-desaturase in the moss Physcomitrella patens. GC-MS analyses demonstrated that P. patens accumulated stigmasterol as the major sterol (56-60% of total sterol) and sitosterol to a lesser extent (8-12%); this sterol profile contrasts with those in higher plants accumulating stigmasterol as a minor component. Recombinant CYP710A13 and CYP710A14 proteins prepared using a baculovirus/insect cell system exhibited the C22-desaturase activity with beta-sitosterol to produce stigmasterol, while campesterol and 24-epi-campesterol were not accepted as the substrates. The K (m) values for beta-sitosterol of CYP710A13 (1.0 +/- A 0.043 mu M) and CYP710A14 (2.1 +/- A 0.17 mu M) were at comparable levels of those reported with higher plant CYP710A proteins. In Arabidopsis T87 cells over-expressing CYP710A14, stigmasterol contents reached a level 20- to 72-fold higher than those in the basal level of T87 cells, confirming the C22-desaturase activity of this P450 enzyme. The occurrence of the end-products together with the enzymes involved in the last step of the pathway substantiated the presence of an entire sterol biosynthetic pathway in P. patens, providing evidence for the conservation of the sterol biosynthetic pathway through the evolutionary process of land plants.