Cloning and Expression of Cytochrome P450 Enzymes Catalyzing the Conversion of Tyrosine to p-Hydroxyphenylacetaldoxime in the Biosynthesis of Cyanogenic Glucosides in Triglochin maritima

Two cDNA clones encoding cytochrome P450 enzymes belonging to the CYP79 family have been isolated from Triglochin maritima. The two proteins show 94% sequence identity and have been designated CYP79E1 and CYP79E2. Heterologous expression of the native and the truncated forms of the two clones in Escherichia coli demonstrated that both encode multifunctional N-hydroxylases catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the two cyanogenic glucosides taxiphyllin and triglochinin in T. maritima. This renders CYP79E functionally identical to CYP79A1 from Sorghum bicolor, and unambiguously demonstrates that cyanogenic glucoside biosynthesis in T. maritima and S. bicolor is catalyzed by analogous enzyme systems with p-hydroxyphenylacetaldoxime as a free intermediate. This is in contrast to earlier reports stipulating p-hydroxyphenylacetonitrile as the only free intermediate in T. maritima. l-3,4-Dihydroxyphenyl[3-(14)C]Ala (DOPA) was not metabolized by CYP79E1, indicating that hydroxylation of the phenol ring at the meta position, as required for triglochinin formation, takes place at a later stage. In S. bicolor, CYP71E1 catalyzes the subsequent conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile. When CYP79E1 from T. maritima was reconstituted with CYP71E1 and NADPH-cytochrome P450 oxidoreductase from S. bicolor, efficient conversion of tyrosine to p-hydroxymandelonitrile was observed.

从海韭（Triglochin maritima）中分离得到两个编码属于CYP79家族的细胞色素P450（cytochrome P450）酶的cDNA克隆。这两个蛋白质的序列同源性达94%，已被分别命名为CYP79E1和CYP79E2。将这两个克隆的天然形式与截短形式在大肠杆菌（Escherichia coli）中进行异源表达，结果证实二者均编码多功能N-羟化酶，可催化酪氨酸转化为对羟基苯乙醛肟，该反应参与了海韭（T. maritima）中两种生氰糖苷（cyanogenic glucosides）紫杉叶苷（taxiphyllin）与海韭苷（triglochinin）的生物合成过程。该结果表明CYP79E在功能上与高粱（Sorghum bicolor）来源的CYP79A1完全一致，并明确证实海韭（T. maritima）与高粱（S. bicolor）的生氰糖苷生物合成均由以对羟基苯乙醛肟作为游离中间体的类似酶系统所催化。这与此前认为对羟基苯乙腈是海韭（T. maritima）中唯一游离中间体的早期研究报道相悖。CYP79E1无法代谢l-3,4-二羟基苯丙氨酸[3-(14)C]（DOPA），这表明合成海韭苷所需的苯环间位羟基化反应发生在后续步骤。在高粱（S. bicolor）中，CYP71E1可催化对羟基苯乙醛肟进一步转化为对羟基扁桃腈。当将海韭（T. maritima）来源的CYP79E1与高粱（S. bicolor）来源的CYP71E1和NADPH-细胞色素P450氧化还原酶（NADPH-cytochrome P450 oxidoreductase）进行体外重构时，可观测到酪氨酸被高效转化为对羟基扁桃腈。