CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzymes metabolizing oximes in the biosynthesis of glucosinolates in Arabidopsis
CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzymes metabolizing oximes in the biosynthesis of glucosinolates in Arabidopsis
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DOI：
10.1104/pp.102.019240
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发表时间：
2003-09-01
期刊：
影响因子：
7.4
通讯作者：
Halkier, BA
中科院分区：
文献类型：
--
作者：
Naur, P;Petersen, BL;Halkier, BA
在芥子油苷途径中，已提出肟后酶对侧链具有低特异性，而对官能团具有高特异性。在这里，我们提供了生化证据的功能作用的两个细胞色素P450，CYP 83 A1和CYP 83 B1，从拟南芥在肟代谢的芥子油苷的生物合成。在酵母（酿酒酵母）中异源表达的重组酶的底物特异性的详细分析中，我们表明，脂肪族肟衍生自链延长的蛋氨酸同系物有效地代谢CYP 83 A1，而CYP 83 B1代谢这些底物的效率非常低。来自苯丙氨酸、色氨酸和酪氨酸的芳香族肟被两种酶代谢，尽管CYP 83 B1对这些底物的亲和力高于CYP 83 A1，特别是在吲哚-3-乙醛肟的情况下，其中K-m值相差50倍。数据表明，CYP 83 A1和CYP 83 B1在植物生理正常条件下是非冗余酶。CYP 83 A1代谢芳香族肟的能力，虽然在小的水平，解释了在不同的发育阶段的CYP 83 B1敲除突变体，rnt 1 -1的吲哚芥子油苷在不同的水平存在。过表达CYP 83 B1的植物含有来自蛋氨酸同系物的脂肪族芥子油苷水平升高，而吲哚芥子油苷的水平在过表达系中几乎是恒定的。连同先前参与肟生产的CYP 79家族成员的表征，这项工作提供了一个框架，芥子油苷的代谢工程和芥子油苷途径的进一步解剖。
In the glucosinolate pathway, the postoxime enzymes have been proposed to have low specificity for the side chain and high specificity for the functional group. Here, we provide biochemical evidence for the functional role of the two cytochromes P450, CYP83A1 and CYP83B1, from Arabidopsis in oxime metabolism in the biosynthesis of glucosinolates. In a detailed analysis of the substrate specificities of the recombinant enzymes heterologously expressed in yeast (Saccharomyces cerevisiae), we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1, whereas CYP83B1 metabolizes these substrates with very low efficiency. Aromatic oximes derived from phenylalanine, tryptophan, and tyrosine are metabolized by both enzymes, although CYP83B1 has higher affinity for these substrates than CYP83A1, particularly in the case of indole-3-acetaldoxime, where there is a 50-fold difference in K-m value. The data show that CYP83A1 and CYP83B1 are nonredundant enzymes under physiologically normal conditions in the plant. The ability of CYP83A1 to metabolize aromatic oximes, albeit at small levels, explains the presence of indole glucosinolates at various levels in different developmental stages of the CYP83B1 knockout mutant, rnt1-1. Plants overexpressing CYP83B1 contain elevated levels of aliphatic glucosinolates derived from methionine homologs, whereas the level of indole glucosinolates is almost constant in the overexpressing lines. Together with the previous characterization of the members of the CYP79 family involved in oxime production, this work provides a framework for metabolic engineering of glucosinolates and for further dissection of the glucosinolate pathway.