Wiles Danielle, Roest James, Shanbhag Bhuvana, Vivian Julian, Beddoe Travis
Department of Ecological, Plant and Animal Science, La Trobe University, Bundoora, Victoria, Australia.
Australian Research Council Research Hub for Medicinal Agriculture, La Trobe University, Bundoora, Victoria, Australia.
PeerJ. 2025 Jul 10;13:e19723. doi: 10.7717/peerj.19723. eCollection 2025.
Terpenoids are the largest and most diverse family of natural products. Volatile terpenes from are crucial in flavours, fragrances, and pharmaceuticals due to their unique odours and biological activities, including antimalarial, antibacterial, and insecticidal properties. Their synthesis is catalysed by terpene synthase (TPS) enzymes, which perform cyclisation and rearrangement reactions of over 55,000 distinct terpene compounds. However, low catalytic efficiency of TPSs limits their use in large-scale commercial production. The complex biochemistry of these reactions is not well understood due to limited enzyme structure information. To address this, we have developed an integrated platform for the systematic expression, purification, enzymatic characterisation, and crystallisation of TPS enzymes from . This workflow combines kinetic, thermostability, and structural analyses with a data-mining-informed crystallisation screen that enabled the production of diffraction-quality crystals. As a demonstration of the platform's utility, ten TPS enzymes were functionally characterised, revealing turnover rates (k) ranging from 0.0011 to 0.0204 s and diverse substrate specificities, with each enzyme producing a distinct product profile, highlighting the need for systematic characterisation of terpene biosynthesis. Our findings provide a framework for the structural and functional study of TPSs. The developed platform sets the stage for future metabolic engineering aimed at optimising terpene production for pharmaceutical, pest management, and synthetic biology applications.
萜类化合物是最大且最多样化的天然产物家族。植物来源的挥发性萜类化合物因其独特气味和生物活性（包括抗疟疾、抗菌和杀虫特性）在香料、香精和制药领域至关重要。它们的合成由萜烯合酶（TPS）催化，这些酶对超过55000种不同的萜类化合物进行环化和重排反应。然而，TPS的低催化效率限制了它们在大规模商业生产中的应用。由于酶结构信息有限，这些反应复杂的生物化学过程尚未得到充分理解。为了解决这一问题，我们开发了一个综合平台，用于系统表达、纯化、酶学表征以及从植物中结晶TPS酶。该工作流程将动力学、热稳定性和结构分析与数据挖掘指导的结晶筛选相结合，从而能够生产出高质量的衍射晶体。作为该平台实用性的证明，对十种植物TPS酶进行了功能表征，结果显示其周转速率（k）在0.0011至0.0204 s之间，底物特异性各异，每种酶都产生独特的产物谱，突出了对植物萜类生物合成进行系统表征的必要性。我们的研究结果为植物TPS的结构和功能研究提供了一个框架。所开发的平台为未来旨在优化萜类化合物生产以用于制药、害虫管理和合成生物学应用的代谢工程奠定了基础。
