Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity
Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity
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菊基二磷酸合酶在植物中作为具有菊酚合酶活性的双功能酶起作用
DOI：
10.1074/jbc.m114.623348
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发表时间：
2014-12-26
影响因子：
4.8
通讯作者：
Jongsma, Maarten A.
中科院分区：
文献类型：
--
作者：
Yang, Ting;Gao, Liping;Jongsma, Maarten A.
菊花二磷酸合成酶(CDS)是除虫菊酯生物合成的第一个途径特异性酶，是应用最广泛的植物源农药。硫化镉催化两个二甲基烯丙基二磷酸(DMAPP)分子的1‘-2-3环丙烷化反应合成菊花二磷酸(CPP)。已知有三种蛋白质催化萜类前体的环丙烷化反应。其中两种是植烯和角鲨烯合成酶，它们同时具有戊烯转移酶和萜烯合成酶的活性。据报道，另一个成员CDS只执行戊烯基转移酶步骤。在这里，我们证明了CDS的NDXXD催化基序，在植物中普遍存在的低底物条件下，也催化了下一步，通过水解二磷酸部分将CPP转化为菊花酚。酶促反应遵循传统的Michaelis-Menten动力学，CPP的Km值为196 mU M。产生菊花酚的半最大活性所需的DMAPP浓度与100µM相似，且在较高的DMAPP浓度下观察到显著的底物抑制。CDS的N端肽被鉴定为一种质体靶向多肽。过量表达CDS的转基因烟草以0.12~0.16微克·h~(-1)鲜重的速率释放菊花。我们建议以DMAPP为底物，将CDS改名为菊花酚合成酶。
Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 mu M. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was similar to 100 mu M, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 mu g h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.