Peters Reuben J, Croteau Rodney B
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
Arch Biochem Biophys. 2003 Sep 15;417(2):203-11. doi: 10.1016/s0003-9861(03)00347-3.
Monoterpene cyclization reactions are initiated by ionization and isomerization of geranyl diphosphate, and proceed, via cyclization of bound linalyl diphosphate, through a series of carbocation intermediates with ultimate termination of the multistep cascade by deprotonation or nucleophile capture. Three structurally and mechanistically related monoterpene cyclases from Salvia officinalis, (+)-sabinene synthase (deprotonation to olefin), 1,8-cineole synthase (water capture), and (+)-bornyl diphosphate synthase (diphosphate capture), were employed to explore the structural determinants of these alternative termination chemistries. Results with chimeric recombinant enzymes, constructed by reciprocally substituting regions of sabinene synthase with the corresponding sequences from bornyl diphosphate synthase or 1,8-cineole synthase, demonstrated that exchange of the C-terminal catalytic domain is sufficient to completely switch the resulting product profile. Exchange of smaller sequence elements identified a region of roughly 70 residues from 1,8-cineole synthase that, when substituted into sabinene synthase, conferred the ability to produce 1,8-cineole. A similar strategy identified a small region of bornyl diphosphate synthase important in conducting the anti-Markovnikov addition to the bornane skeleton. Observations made with these chimeric monoterpene cyclases are discussed in the context of the recently determined crystal structure for bornyl diphosphate synthase.
单萜环化反应由香叶基二磷酸的离子化和异构化引发，通过结合的芳樟基二磷酸的环化，经由一系列碳正离子中间体进行，多步级联反应最终通过去质子化或亲核试剂捕获而终止。使用来自鼠尾草的三种结构和机制相关的单萜环化酶，即（+）-桧烯合酶（去质子化生成烯烃）、1,8-桉叶素合酶（水捕获）和（+）-冰片基二磷酸合酶（二磷酸捕获），来探索这些替代终止化学过程的结构决定因素。通过用冰片基二磷酸合酶或1,8-桉叶素合酶的相应序列相互替换桧烯合酶的区域构建嵌合重组酶，结果表明C末端催化结构域的交换足以完全改变所得产物谱。较小序列元件的交换确定了1,8-桉叶素合酶中大约70个残基的区域，当将该区域替换到桧烯合酶中时，赋予了产生1,8-桉叶素的能力。类似策略确定了冰片基二磷酸合酶中的一个小区域，该区域在对莰烷骨架进行反马氏加成中起重要作用。结合最近确定的冰片基二磷酸合酶晶体结构，讨论了这些嵌合单萜环化酶的观察结果。
