Benveniste I, Tijet N, Adas F, Philipps G, Salaün J P, Durst F
Institut de Biologie Moléculaire des Plantes-CNRS, Département d'Enzymologie Cellulaire et Moléculaire, Strasbourg, France.
Biochem Biophys Res Commun. 1998 Feb 24;243(3):688-93. doi: 10.1006/bbrc.1998.8156.
The A. thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals. One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A. thaliana. This cDNA coded the first member of a new plant P450 family and was termed CYP86A1. The deduced peptide sequence showed highest homology with P450s from families 4 and 52. To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase. Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity. CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane. Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A. thaliana.
利用源自细胞色素P450家族CYP52和CYP4的共有基序筛选拟南芥EST数据库,CYP52和CYP4在念珠菌和哺乳动物中催化脂肪酸和烷烃的ω-羟基化反应。通过这种方式检测到一个EST cDNA片段,并从拟南芥cDNA文库中克隆出相应的全长cDNA。该cDNA编码一个新的植物P450家族的首个成员,被命名为CYP86A1。推导的肽序列与4和52家族的细胞色素P450具有最高同源性。为了确认催化功能,CYP86A1在过表达自身NADPH-细胞色素P450还原酶的酵母中表达。通过分光光度法、SDS-PAGE和催化活性证明了有效表达。发现CYP86A1催化链长从C12到C18的饱和与不饱和脂肪酸的ω-羟基化反应,但不催化十六烷的ω-羟基化反应。通过Southern杂交分析基因组结构表明拟南芥中编码CYP86A1的是单基因。