Bak S, Kahn R A, Nielsen H L, Moller B L, Halkier B A
Department of Plant Biology, Royal Veterinary and Agricultural University, Copenhagen, Denmark.
Plant Mol Biol. 1998 Feb;36(3):393-405. doi: 10.1023/a:1005915507497.
A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450- (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG-was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained. Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH-cytochrome P450-reductase in L-alpha-dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction. CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450-reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.
分离出了一种编码多功能细胞色素P450（CYP71E1）的cDNA，该细胞色素参与高粱（Sorghum bicolor (L.) Moench）中氰基糖苷蜀黍苷的生物合成。采用了一种基于A类细胞色素P450的三个共有序列——(V/I)KEX(L/F)R、FXPERF和PFGXGRRXCXG——的PCR方法。除了获得一个编码高粱肉桂酸4-羟化酶的PCR片段外，还得到了三种新的细胞色素P450（CYP71E1、CYP98和CYP99）。在L-α-二月桂酰磷脂酰胆碱微团中，用在大肠杆菌中异源表达的重组CYP71E1和高粱NADPH-细胞色素P450还原酶进行重组实验，确定CYP71E1是在蜀黍苷生物合成中催化对羟基苯乙醛肟转化为对羟基苯乙腈的细胞色素P450。根据提出的蜀黍苷生物合成途径，CYP71E1催化肟脱水生成相应的腈，随后腈进行C-羟基化生成对羟基苯乙腈。在大肠杆菌细胞中体内给予肟会导致腈的积累，这表明大肠杆菌中的黄素氧还蛋白/黄素氧还蛋白还原酶系统仅能在脱水反应中支持CYP71E1，而不能支持随后的C-羟基化反应。CYP79催化酪氨酸转化为对羟基苯乙醛肟，这是氰基糖苷蜀黍苷生物合成中的第一个关键步骤。将CYP79和CYP71E1与高粱NADPH-细胞色素P450还原酶一起重组，导致酪氨酸转化为对羟基苯乙腈，即氰基糖苷蜀黍苷生物合成途径的膜部分。分离出CYP71E1的cDNA以及先前分离出的CYP79的cDNA，为植物中氰基糖苷水平的组织特异性调控提供了重要工具，以优化食品安全和抗虫害能力。
