Structural investigation of a type III polyketide synthase involved in cannabinoid biosynthesis

参与大麻素生物合成的 III 型聚酮合酶的结构研究

• 批准号: 10387655
• 负责人: Kristofer R. Gonzalez-DeWhitt
• 金额: $ 4.14万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-30 至 2024-06-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387655
• 关键词: Acids; Acyl Coenzyme A; Amino Acids; Anabolism; Architecture; Binding; Biochemical; Biogenesis; COVID-19 pandemic; COVID-19 prevention; Cannabidiol; Cannabinoids; Cannabis; Cannabis sativa plant; Centers for Disease Control and Prevention (U.S.); Coenzyme A; Complex; Crystallization; Crystallography; Cyclization; Data; Data Set; Decarboxylation; Discrimination; Endocannabinoids; Enzymes; Fostering; Freezing; Goals; Guidelines; High Pressure Liquid Chromatography; Human; Hydrolysis; Industry; Investigation; Length; Liquid substance; Malonyl Coenzyme A; Methods; Molecular Conformation; Mutagenesis; Nitrogen; Pathway interactions; Pharmaceutical Chemistry; Pharmacodynamics; Pharmacologic Substance; Pharmacology; Physical condensation; Plants; Process; Production; Proteins; Reaction; Recommendation; Research; Resolution; Roentgen Rays; Series; Statutes and Laws; Structure; Substrate Specificity; Tetrahydrocannabinol; Therapeutic; Time; Workplace; acyl group; cannabigerolic acid; data quality; experimental study; hexanoyl-coenzyme A; improved; insight; interest; liquid chromatography mass spectrometry; mutant; novel; oxidation; pandemic disease; phytocannabinoid; polyketide synthase; premature; working group

PROJECT SUMMARY Phytocannabinoids from Cannabis sativa L. are natural plant products with therapeutic potential in humans. Rapidly changing Cannabis legislation has fostered a burgeoning pharmaceutical cannabinoid industry, which is projected to be worth USD 5.7 billion in 2027. To keep pace with rising demand, research groups are working to develop heterologous expression methods for cannabinoid production. Current strategies show promise but suffer from low product yields. In this biosynthetic pathway, a plant type III polyketide synthase referred to as tetraketide synthase (TKS) catalyzes a highly modular reaction to generate essential precursors in the biogenesis of more than 113 phytocannabinoids, such as cannabidiol and Δ9-tetrahydrocannabinol. The reaction is prone to forming premature hydrolysis by- products derailing phytocannabinoid biosynthesis. The objective of this application is to establish a structural and mechanistic basis for understanding the TKS reaction. Preliminary experiments employing X-ray protein crystallography, structure-guided mutagenesis, and high performance liquid chromatography provide critical insight into this highly modular reaction. A method to generate catalytically active TKS crystals was developed. The resulting crystal structures provide a mechanistically relevant view of the TKS substrate-binding pocket. This is the first example of such high-resolution structural information being obtained for TKS. The proposed research strategy builds upon these preliminary experiments to characterize the architecture of the TKS substrate-binding pocket to identify key residues regulating substrate discrimination and polyketide formation (Aim 1). Time-resolved X-ray protein crystallography experiments will be pursued to investigate the structural and conformational changes occurring within the TKS substrate-binding pocket during polyketide elongation (Aim 2). Completion of these proposed aims will provide much needed structural insight elucidating the mechanism of multiple substrate discrimination and production formation in the TKS-catalyzed reaction. The results will have broad implications for enabling biosynthetic approaches to cannabinoid production.

项目总结 大麻中的植物大麻素是一种天然植物产品，具有治疗多种疾病的潜力。 人类。迅速变化的大麻立法催生了一种迅速发展的药用大麻 工业，预计到2027年将达到57亿美元。为了跟上不断增长的需求， 研究小组正在努力开发生产大麻素的异源表达方法。 目前的战略显示出了希望，但产品收益率较低。在这个生物合成途径中，一种植物 被称为四酮合成酶(TKS)的III型聚酮合成酶催化一种高度模块化的反应 在113种以上植物大麻素的生物发生过程中产生必要的前体，例如 大麻二酚和Δ9-四氢大麻酚。该反应容易通过以下方式形成过早的水解- 使植物大麻素生物合成脱轨的产品。此应用程序的目标是建立一个 理解TKS反应的结构和机理基础。初步实验采用 X射线蛋白质结晶学、结构导向诱变和高效液体 层析为这种高度模块化的反应提供了关键的洞察力。一种生成数据的方法 制备了具有催化活性的TKS晶体。由此产生的晶体结构提供了一种机械上的 相关查看TKS衬底绑定口袋。这是第一个这样高分辨率的例子 正在为将军澳获得结构信息。拟议的研究战略建立在这些基础上 初步实验表征TKS底物结合口袋的结构以识别 调节底物辨别和聚酮形成的关键残留物(目标1)。时间分辨X射线 蛋白质结晶学实验将被用来研究结构和构象。 聚酮延伸过程中TKS底物结合袋内发生的变化(目标2)。 这些拟议目标的完成将提供亟需的结构性洞察，以澄清 TKS催化反应中多底物辨别和产物形成的机理。 这一结果将对生物合成方法生产大麻素产生广泛的影响。