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: 10643698
• 负责人: Kristofer R. Gonzalez-DeWhitt
• 金额: $ 4.27万
• 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/10643698
• 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.); Classification; Coenzyme A; Complex; Crystallography; Cyclization; Data; Data Set; Decarboxylation; Discrimination; 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; Pharmacologic Substance; Physical condensation; Plant Extracts; Plants; Process; Production; Property; Proteins; Reaction; Recommendation; Research; Resolution; Roentgen Rays; Series; Statutes and Laws; Structure; Substrate Specificity; Tetrahydrocannabinol; Therapeutic; Time; Visualization; Workplace; acyl group; cannabigerolic acid; current pandemic; data quality; endogenous cannabinoid system; experimental study; hexanoyl-coenzyme A; improved; insight; interest; liquid chromatography mass spectrometry; mutant; novel; oxidation; pharmacokinetics and pharmacodynamics; pharmacologic; phytocannabinoid; polyketide synthase; polyketides; premature; secondary metabolite; 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.

项目摘要 大麻中的植物大麻素是天然植物产品，具有治疗潜力， 人类快速变化的大麻立法促进了一种新兴的药用大麻素 该行业预计在2027年价值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用于大麻素生产的异源表达方法。 目前的战略显示出希望，但产品产量低。在这个生物合成途径中， III型聚酮合酶称为四酮合酶（TKS），催化高度模块化的反应 在超过113种植物大麻素的生物发生中产生必需的前体，例如 大麻二酚和Δ9-四氢大麻酚。该反应易于形成过早的水解- 使植物大麻素生物合成脱轨的产物。本申请的目的是建立一个 理解TKS反应的结构和机理基础。初步实验采用 X射线蛋白质晶体学、结构导向突变和高效液相色谱 色谱提供了对这种高度模块化反应的关键洞察。一种生成 开发了催化活性TKS晶体。所得的晶体结构提供了机械上的 TKS底物结合口袋的相关视图。这是第一个如此高分辨率的例子 获取TKS的结构信息。拟议的研究战略建立在这些基础之上 初步实验来表征TKS底物结合口袋的结构，以鉴定 调节底物识别和聚酮化合物形成的关键残基（目的1）。时间分辨X射线 蛋白质晶体学实验将进行研究的结构和构象 在聚酮化合物延伸过程中TKS底物结合口袋内发生的变化（目的2）。 这些拟议目标的完成将提供急需的结构性见解， TKS催化反应中多底物识别和产物形成的机制。 这些结果将对大麻素生产的生物合成方法产生广泛的影响。