Arabidopsis CYP707As encode (+)-abscisic acid 8′-hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid
Arabidopsis CYP707As encode (+)-abscisic acid 8′-hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid
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DOI：
10.1104/pp.103.037614
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发表时间：
2004-04-01
期刊：
影响因子：
7.4
通讯作者：
Mizutani, M
中科院分区：
文献类型：
--
作者：
Saito, S;Hirai, N;Mizutani, M
脱落酸（ABA）参与了正常生长发育的许多关键过程以及对环境胁迫的适应性反应。为了正确和准确的行动，生理上活跃的ABA水平是通过微调从头生物合成和分解代谢来控制的。ABA 8′位羟基化是ABA分解代谢的关键步骤，该反应由ABA 8′-羟化酶（一种细胞色素P450）催化。在这里，我们证明CYP707As是负责(+)-ABA的8'-羟基化的P450。首先，从拟南芥中克隆了所有四个CYP707A cdna，并利用杆状病毒系统在昆虫细胞中生产重组蛋白。表达CYP707A3的昆虫细胞有效地代谢(+)-ABA生成相酸，即8′-羟基aba的异构化形式。昆虫大提琴微粒体具有很强的(+)-ABA 8′-羟基化活性(k -m = 1.3 muM, k（cat) = 15 min(-1)）。溶解后的CYP707A3蛋白结合(+)- aba，结合常数K-s = 3.5 muM，不结合(-)- aba。对反应产物的详细分析证实CYP707A3不具有8′-羟基- aba对相酸的异构化活性。进一步实验发现拟南芥CYP707A1和CYP707A4也编码ABA 8′-羟化酶。CYP707A基因的转录本在盐、渗透和脱水胁迫以及ABA的作用下增加。这些结果表明CYP707A家族通过(+)-ABA的8′-羟基化作用在调节ABA水平中起关键作用。
Abscisic acid (ABA) is involved in a number of critical processes in normal growth and development as well as in adaptive responses to environmental stresses. For correct and accurate actions, a physiologically active ABA level is controlled through fine-tuning of de novo biosynthesis and catabolism. The hydroxylation at the 8'-position of ABA is known as the key step of ABA catabolism, and this reaction is catalyzed by ABA 8'-hydroxylase, a cytochrome P450. Here, we demonstrate CYP707As as the P450 responsible for the 8'-hydroxylation of (+)-ABA. First, all four CYP707A cDNAs were cloned from Arabidopsis and used for the production of the recombinant proteins in insect cells using a baculovirus system. The insect cells expressing CYP707A3 efficiently metabolized (+)-ABA to yield phaseic acid, the isomerized form of 8'-hydroxy-ABA. The microsomes from the insect cello exhibited very strong activity of 8'-hydroxylation of (+)-ABA (K-m = 1.3 muM and k(cat) = 15 min(-1)). The solubilized CYP707A3 protein bound (+)-ABA with the binding constant K-s = 3.5 muM, but did not bind (-)-ABA. Detailed analyses of the reaction products confirmed that CYP707A3 does not have the isomerization activity of 8'-hydroxy-ABA to phaseic acid. Further experiments revealed that Arabidopsis CYP707A1 and CYP707A4 also encode ABA 8'-hydroxylase. The transcripts of the CYP707A genes increased in response to salt, osmotic, and dehydration stresses as well as ABA. These results establish that the CYP707A family plays a key role in regulating the ABA level through the 8'-hydroxylation of (+)-ABA.