CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis
CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis
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DOI：
10.1046/j.1365-313x.2003.01679.x
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发表时间：
2003-03-01
期刊：
影响因子：
7.2
通讯作者：
Halkier, BA
中科院分区：
文献类型：
--
作者：
Chen, SX;Glawischnig, E;Halkier, BA
CYP79家族的细胞色素P450在芥子油苷的生物合成中催化氨基酸转化为肟，芥子油苷是一组已知参与植物防御的天然植物产物，并且是风味化合物、癌症预防剂和生物除草剂的来源。我们报告了一个详细的生化分析的底物特异性和动力学的CYP79F1和CYP79F2，两个细胞色素P450参与的脂肪族芥子油苷在拟南芥的生物合成。使用重组CYP79F1和CYP79F2在大肠杆菌和酿酒酵母中表达，分别，我们表明，CYP79F1代谢单到六高蛋氨酸，导致短链和长链脂肪族硫代葡萄糖苷。相反，CYP79F2仅代谢长链延长的五和六高蛋氨酸。CYP79F1和CYP79F2在空间和发育上受到调节，具有不同的基因表达模式。CYP79F2在下胚轴和根中高度表达，而CYP79F1在子叶、莲座叶、茎和长角果中强烈表达。转座子标记的CYP79F1敲除突变体完全缺乏短链脂肪族芥子油苷，但具有增加的长链脂肪族芥子油苷水平，特别是在叶和种子中。转座子标记的CYP79F2敲除突变体中长链脂肪族芥子油苷的水平显著降低，而短链脂肪族芥子油苷的水平不受影响。CYP79F1和CYP79F2的生物化学表征，基因表达分析，结合敲除突变体的硫代葡萄糖苷谱，证明了这些酶的功能作用。这为导致脂肪族硫代葡萄糖苷生物合成的代谢网络以及改变脂肪族硫代葡萄糖苷概况以提高营养价值和抗害虫能力的代谢工程提供了有价值的见解。
Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana . Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.