Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29, Yoshida-Shimoadachi, Sakyo-ku, Kyoto 606-8501, Japan.
Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29, Yoshida-Shimoadachi, Sakyo-ku, Kyoto 606-8501, Japan.
Phytochemistry. 2014 Jun;102:46-54. doi: 10.1016/j.phytochem.2014.03.020. Epub 2014 Apr 8.
Geraniol and linalool are acyclic monoterpenes found in plant essential oils that have attracted much attention for their commercial use and in pharmaceutical studies. They are synthesized from geranyl diphosphate (GDP) by geraniol and linalool synthases, respectively. Both synthases are very similar at the amino acid level and share the same substrate; however, the position of the GDP to which they introduce hydroxyl groups is different. In this study, the mechanisms underlying the regiospecific hydroxylation of geraniol and linalool synthases were investigated using a domain swapping approach and site-directed mutagenesis in perilla. Sequences of the synthases were divided into ten domains (domains I to IV-4), and each corresponding domain was exchanged between both enzymes. It was shown that different regions were important for the formation of geraniol and linalool, namely, domains IV-1 and -4 for geraniol, and domains III-b, III-d, and IV-4 for linalool. These results suggested that the conformation of carbocation intermediates and their electron localization were seemingly to be different between geraniol and linalool synthases. Further, five amino acids in domain IV-4 were apparently indispensable for the formation of geraniol and linalool. According to three-dimensional structural models of the synthases, these five residues seemed to be responsible for the different spatial arrangement of the amino acid at H524 in the case of geraniol synthase, while N526 is the corresponding residue in linalool synthase. These results suggested that the side-chains of these five amino acids, in combination with several relevant domains, localized the positive charge in the carbocation intermediate to determine the position of the introduced hydroxyl group.
香叶醇和芳樟醇是植物精油中的环状单萜，由于其商业用途和在药物研究中的重要性而备受关注。它们分别由香叶基二磷酸（GDP）合成香叶醇合酶和芳樟醇合酶合成。两种合酶在氨基酸水平上非常相似，并且具有相同的底物；然而，它们引入羟基的 GDP 位置不同。在这项研究中，使用域交换方法和定向突变在紫苏中研究了香叶醇和芳樟醇合酶的区域特异性羟化作用的机制。合酶的序列分为十个结构域（结构域 I 至 IV-4），并且两个酶之间交换了每个相应的结构域。结果表明，不同的区域对于香叶醇和芳樟醇的形成很重要，即结构域 IV-1 和 -4 对于香叶醇，以及结构域 III-b、III-d 和 IV-4 对于芳樟醇。这些结果表明，碳正离子中间体的构象及其电子定位在香叶醇合酶和芳樟醇合酶之间似乎不同。此外，结构域 IV-4 中的五个氨基酸显然对于香叶醇和芳樟醇的形成是不可缺少的。根据合酶的三维结构模型，这五个残基似乎负责香叶醇合酶中 H524 处氨基酸的不同空间排列，而 N526 是芳樟醇合酶中的相应残基。这些结果表明，这五个氨基酸的侧链与几个相关的结构域结合，将正电荷定位在碳正离子中间体中，以确定引入的羟基的位置。
