Truncation of limonene synthase preprotein provides a fully active 'Pseudomature' form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair
Truncation of limonene synthase preprotein provides a fully active 'Pseudomature' form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair
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DOI：
10.1021/bi980854k
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发表时间：
1998-09-01
期刊：
影响因子：
2.9
通讯作者：
Croteau, R
中科院分区：
文献类型：
--
作者：
Williams, DC;McGarvey, DJ;Croteau, R
单萜环化酶柠檬烯合成酶将香叶二磷酸转化为单环烯烃，构成了萜类环化酶催化的最简单模型。绿薄荷(-)- 4s -柠檬烯合成酶前蛋白具有较长的质体靶向序列。由于宿主密码子的使用、包涵体的形成以及细菌伴侣蛋白与转运肽的紧密关联，在大肠杆菌中表达全长前蛋白遇到了困难。相对于在体内通过在质体中进行蛋白水解加工而产生的n阻断天然蛋白的混合物，纯化的前蛋白在动力学上也受到损害。因此，在单萜合成酶中高度保守的串联精氨酸对（R58R59）之前的靶向序列被移除。这种截断蛋白的表达，来自一个编码两个罕见精氨酸密码子（pSBET）的tRNA的载体，提供了一种可溶性的、可处理的“假成熟”形式的酶，其催化效率比本地物种更高。截断至R58或替换R59，所产生的酶不能将天然底物二磷酸香叶基通过酶催化形成的叔烯丙基异构体3s -二磷酸芳樟烯转化为(-)-柠檬烯。然而，这些酶能够将外源供应的3s -芳樟二磷酸环化成烯烃产物。这一结果表明，串联精氨酸在伴随中间3s -芳樟二磷酸形成的独特的二磷酸迁移步骤中起作用，并在单萜合成酶催化的最终环化反应之前起作用。基于倍半萜环化酶表马圈子烯合成酶的三维结构，通过(-)- 4s -柠檬烯合成酶的同源性建模，可以推断出这一耦合异构化-环化反应序列的结构基础。
The monoterpene cyclase limonene synthase transforms geranyl diphosphate to a monocyclic olefin and constitutes the simplest model for terpenoid cyclase catalysis. (-)-4S-Limonene synthase preprotein from spearmint bears a long plastidial targeting sequence. Difficulty expressing the full-length preprotein in Escherichia coli is encountered because of host codon usage, inclusion body formation, and the tight association of bacterial chaperones with the transit peptide. The purified preprotein is also kinetically impaired relative to the mixture of N-blocked native proteins produced in vivo by proteolytic processing in plastids. Therefore, the targeting sequence, that precedes a tandem pair of arginines (R58R59) which is highly conserved in the monoterpene synthases, was removed. Expression of this truncated protein, from a vector that encodes a tRNA for two rare arginine codons (pSBET), affords a soluble, tractable 'pseudomature' form of the enzyme that is catalytically more efficient than the native species. Truncation up to and including R58, or substitution of R59, yields enzymes that are incapable of converting the natural substrate geranyl diphosphate, via the enzymatically formed tertiary allylic isomer 3S-linalyl diphosphate, to (-)-limonene. However, these enzymes are able to cyclize exogenously supplied 3S-linalyl diphosphate to the olefinic product. This result indicates a role for the tandem arginines in the unique diphosphate migration step accompanying formation of the intermediate 3S-linalyl diphosphate and preceding the final cyclization reaction catalyzed by the monoterpene synthases. The structural basis for this coupled isomerization-cyclization reaction sequence can be inferred by homology modeling of (-)-4S-limonene synthase based on the three-dimensional structure of the sesquiterpene cyclase epiaristolochene synthase.