Wiles Danielle, Roest James, Vivian Julian P, Beddoe Travis
ARC Research Hub for Medicinal Agriculture, La Trobe University, Bundoora 3083, Australia; Department of Animal, Plant, and Soil Science, La Trobe University, Bundoora 3083, Australia.
St. Vincents Institute Medical Research, Fitzroy, Victoria, Australia.
J Struct Biol. 2025 Sep;217(3):108227. doi: 10.1016/j.jsb.2025.108227. Epub 2025 Jun 18.
Cannabis sativa is a high-value plant renowned for its diverse chemical composition and abundant terpene content, contributing to its unique aroma, flavour, and therapeutic effects. Terpenes significantly influence consumer preference for C. sativa products, driving scientific interest in optimising terpene expression profiles and shaping the selective breeding of terpene profiles in C. sativa cultivars. In particular, the monoterpene, terpinolene, is influential in defining the sensory and therapeutic qualities of many C. sativa strains due to its woody, citrus-like aroma. Here we report the 2.5 Å resolution crystal structure of terpinolene synthase (CsTOS) from C. sativa in its apo form. The structure exhibits the class I monoterpene synthase fold with an open active site conformation. Using site-directed mutagenesis, we identified H618 as a key residues in determining product specificity. Substituting H618 with charged residues resulted in the preferential formation of limonene over terpinolene, highlighting its critical role in stabilising the substrate intermediate. Additionally, novel mutations uncovered an extended epistatic network of residues within 5 Å of the active site, spanning the α-helical bundle of the terpene synthase fold. These interactions contribute to monoterpene formation by modulating substrate positioning and catalytic activity. These insights advance our understanding of monoterpene biosynthesis and enable the targeted engineering of terpene synthases for customised terpene production, offering significant potential for the C. sativa industry.
大麻是一种高价值植物，以其多样的化学成分和丰富的萜烯含量而闻名，这赋予了它独特的香气、风味和治疗效果。萜烯显著影响消费者对大麻产品的偏好，激发了科学界对优化萜烯表达谱以及塑造大麻品种中萜烯谱选择性育种的兴趣。特别是单萜萜品油烯，由于其木质、类似柑橘的香气，在定义许多大麻菌株的感官和治疗品质方面具有重要影响。在此，我们报告了来自大麻的萜品油烯合酶（CsTOS）无辅基形式的2.5埃分辨率晶体结构。该结构呈现出具有开放活性位点构象的I类单萜合酶折叠。通过定点诱变，我们确定H618是决定产物特异性的关键残基。用带电荷的残基取代H618导致柠檬烯比萜品油烯优先形成，突出了其在稳定底物中间体中的关键作用。此外，新的突变揭示了活性位点5埃范围内的一个扩展的上位残基网络，跨越萜烯合酶折叠的α螺旋束。这些相互作用通过调节底物定位和催化活性促进单萜的形成。这些见解增进了我们对单萜生物合成的理解，并使萜烯合酶的靶向工程能够用于定制萜烯生产，为大麻产业提供了巨大潜力。
