Molecular cloning and characterization of a new linalool synthase
Molecular cloning and characterization of a new linalool synthase
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DOI：
10.1016/s0003-9861(02)00348-x
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发表时间：
2002-09-01
影响因子：
3.9
通讯作者：
Croteau, R
中科院分区：
文献类型：
--
作者：
Crowell, AL;Williams, DC;Croteau, R
薄荷香。（佛手柑薄荷：Lamiaceae）产生一种仅含有无环单萜醇(-)- 3r -芳樟醇及其醋酸酯的精油。基于单萜烯合成酶与该植物家族的单萜烯环化酶序列相似的假设，克隆策略产生了编码(-)- 3r -芳樟醇合成酶的cDNA。该单萜合成酶的核苷酸序列与薄荷（Lamiaceac）家族的几个单萜环化酶相似（62-72%，同源），但与克拉氏菌的3s -芳樟醇合成酶（同源41%，该复合基因似乎是最近起源的）和青蒿的3r -芳樟醇合成酶（同源52%，该基因的功能作用不确定）有很大差异。在大肠杆菌中异种表达cDNA的截短版本（其中plastidial传递肽被删除）允许酶的纯化和表征，该酶被证明具有与其他已知单萜环化酶相似的大多数特性，但天然底物二磷酸香叶基的k -m值为56 muM， k（cat）为0.83 s（-1）。该3r -芳樟醇合成酶的动力学常数高于任何已定义的单萜环化酶，但动力学效率不接近克氏菌的3s -芳樟醇合成酶。虽然二磷酸芳樟醇是单萜环化酶反应的酶结合中间体，但香叶基底物的叔烯丙基异构体并不是与柠檬茅合成酶结合的芳樟醇的有效前体。对这种来自薄荷的芳樟醇合成酶的活性位点进行建模，并与系统发育相关的单萜环化酶的活性位点进行比较，结果显示，在启动亲电反应序列的电离步骤的二磷酸部分的结合以及水进入活性位点以允许初始形成的碳阳离子中间体的立体选择性猝灭以产生3r -芳樟醇方面存在结构差异。(C) 2002 Elsevier Science （USA）。所有灯光保留。
Mentha citrata Ehrh. (bergamot mint: Lamiaceae) produces an essential oil containing only the acyclic monoterpenol (-)-3R-linalool and its acetate ester. A cloning strategy based upon the assumption that the responsible monoterpene synthase would resemble,, in sequence, monoterpene cyclases from this plant family yielded a cDNA encoding the (-)-3R-linalool synthase. The nucleotide sequence of this monoterpene synthase is similar to those of several monoterpene cyclases from the mint (Lamiaceac) family (62-72%, identity), but differs substantially from that of 3S-linalool synthase from Clarkia (41% identity; this composite gene appears to be of recent origin) and from that of 3R-linalool synthase from Artemisia (52% identity; the functional role of this gene is uncertain). Heterologous expression in Escherichia coli of a truncated version of the cDNA (in which the plastidial transit peptide was deleted) allowed purification and characterization of the enzyme, which was shown to possess most properties similar to other known monoterpene cyclases, but with a K-m value for the natural substrate, geranyl diphosphate, of 56 muM with k(cat) of 0.83 s(-1). These kinetic constants for this 3R-linalool synthase are higher than those of any defined monoterpene cyclase, but the kinetic efficiency does not approach that reported for the 3S-linalool synthase from Clarkia. Although linalyl diphosphate is an enzyme-bound intermediate of monoterpene cyclase reactions, this tertiary allylic isomer of the geranyl substrate is, not an efficient precursor of linalool with the M. citrata synthase. Modeling of the active site of this linalool synthase from Mentha and comparison to the modeled active sites of phylogenetically related monoterpene cyclases revealed structural differences in the binding of the diphosphate moiety which initiates the ionization step of the electrophilic reaction sequence and in the access of water to the active site to permit stereoselective quenching of the initially formed carbocationic intermediate to produce 3R-linalool. (C) 2002 Elsevier Science (USA). All Lights reserved.