Structure and Function of Biosynthetic Enzymes

生物合成酶的结构和功能

• 批准号: 8133333
• 负责人: DAVID W CHRISTIANSON
• 金额: $ 31.96万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8133333
• 关键词: Accounting; Active Sites; Anabolism; Analgesics; Anti-Bacterial Agents; Anti-Inflammatory Agents; Anti-inflammatory; Antifungal Agents; Antifungal Antibiotics; Antineoplastic Agents; Architecture; Biological; Biological Assay; Biological Factors; Biology; C-terminal; Carbon; Characteristics; Chemicals; Chemistry; Complex; Cyclization; Diphosphates; Dissection; Diterpenes; Engineering; Enzymatic Biochemistry; Enzymes; Equilibrium; Evolution; Exhibits; Family; Foundations; Funding; Generations; Genus Mentha; Health; Human; Individual; Kinetics; Lead; Malignant Neoplasms; Mass Fragmentography; Menthol; Metabolic Pathway; Metabolism; Metals; Molecular Chaperones; Molecular Conformation; Mouse-ear Cress; N-terminal; Natural Products Chemistry; Nature; Plant Roots; Play; Property; Protein Engineering; Reaction; Reporting; Roentgen Rays; Role; Sesquiterpenes; Site; Specificity; Staging; Structure; Structure-Activity Relationship; Synthesis Chemistry; Terpenes; Terpenoid Biosynthesis Pathway; Time; Variant; Work; X-Ray Crystallography; aqueous; aristolochene synthase; base; biosynthetic product; chemical bond; chemical reaction; chemotherapeutic agent; cholesterol biosynthesis; delta-cadinene synthase; drug discovery; ent-kaurene synthetase A; farnesyl pyrophosphate; fascinate; geosmin; ionization; isoprenoid; mutant; novel; pi bond; programs; protein structure; protonation; public health relevance; research study; scaffold; stereochemistry; structural biology; synthetic biology; terpene synthase; three dimensional structure; trichodiene synthase

DESCRIPTION (provided by applicant): Thousands of terpenoid derivatives found throughout Nature are involved in diverse biosynthetic and metabolic pathways such as cholesterol biosynthesis in humans and menthol biosynthesis in mint. Notably, many terpenoids have been used as medicinal agents since the times of antiquity due to their analgesic, antibiotic, and antifungal properties. In spite of the universal importance of this family of natural products for human health, the three-dimensional structures of terpenoid cyclases have only been reported relatively recently; the majority of these structure determinations have been supported by GM56838. Terpenoid cyclases (a.k.a. terpene synthases) catalyze the cyclization of a common allylic pyrophosphate substrate, such as farnesyl diphosphate, to form one of hundreds of possible products. The terpenoid cyclase plays a critical role as a template in chaperoning substrate and intermediate conformations. The cyclization reaction can be very specific and lead to the formation of one exclusive product, or it can be somewhat promiscuous and lead to the formation of several products. Thus, the terpenoid cyclases comprise an exciting class of biosynthetic enzymes from both the biological and the chemical perspectives. In the current funding period, we have determined the X-ray crystal structures of the sesquiterpene cyclases A. terreus aristolochene synthase, delta-cadinene synthase, and epi-isozizaene synthase; we have established the structural basis for aberrant product formation by wild-type trichodiene synthase and its site-specific mutants; and we have generated the first crystals of a diterpene cyclase, copalyl diphosphate synthase. We aim to build upon this outstanding structural foundation in the next funding period by dissecting detailed structure-function relationships in aristolochene synthase and epi-isozizaene synthase to better understand the structural basis of biosynthetic diversity. Specifically, we will study site- specific variants engineered to generate alternative products, and we will develop a structure-based approach for generating new cyclic terpenoids in protein engineering experiments. Additionally, we will determine the X-ray crystal structures of copalyl diphosphate synthase and geosmin synthase to explore the evolution of domain architecture in multidomain terpenoid cyclases. These studies will illuminate the evolutionary roots of biosynthetic diversity in the greater family of terpenoid synthases. PUBLIC HEALTH RELEVANCE: Structural and functional studies of the terpenoid cyclases show how these novel enzymes generate the largest and most diverse family of natural products found on the Earth. Importantly, many terpenoids exhibit useful medicinal properties, e.g., as antibacterial, antifungal, anti-inflammatory, or anticancer agents. Therefore, understanding and engineering terpenoid cyclase function in generating complex carbon scaffolds with great specificity and efficiency will ultimately enable drug discovery at the interface of synthetic chemistry and synthetic biology.

描述（由申请人提供）：在整个自然界中发现的数千种萜类衍生物涉及多种生物合成和代谢途径，例如人类中的胆固醇生物合成和薄荷中的薄荷醇生物合成。值得注意的是，许多萜类化合物由于其镇痛、抗生素和抗真菌特性，自古代以来一直被用作药剂。尽管这个家族的天然产物对人类健康的普遍重要性，萜类化合物环化酶的三维结构只是最近才被报道;这些结构测定的大部分已经由GM 56838支持。萜类环化酶（也称为萜烯脱氢酶）催化常见的烯丙基焦磷酸底物如法呢基二磷酸的环化，形成数百种可能产物中的一种。萜类环化酶作为模板在陪伴底物和中间构象中起着关键作用。环化反应可以是非常特异性的，导致形成一种唯一的产物，或者它可以是有点混杂的，导致形成几种产物。因此，萜类化合物环化酶从生物学和化学的角度都包括一类令人兴奋的生物合成酶。在当前的资助期内，我们已经确定了倍半萜环化酶A的X射线晶体结构。土马兜铃烯合酶、δ-杜松烯合酶和表-异齐烯合酶;我们已经建立了野生型异齐烯合酶及其位点特异性突变体形成异常产物的结构基础;并且我们已经产生了二萜环化酶柯巴基二磷酸合酶的第一个晶体。我们的目标是在下一个资助期内，通过解剖马兜铃烯合酶和epi-isozizaene合酶的详细结构-功能关系，以更好地了解生物合成多样性的结构基础。具体来说，我们将研究特定位点的变异体，这些变异体被工程化以产生替代产品，我们将开发一种基于结构的方法，用于在蛋白质工程实验中产生新的环状萜类化合物。此外，我们将确定柯巴基二磷酸合酶和土臭素合酶的X-射线晶体结构，以探索多结构域萜类化合物环化酶结构域的进化。这些研究将阐明萜类化合物脱氢酶大家族生物合成多样性的进化根源。 公共卫生关系：萜类环化酶的结构和功能研究显示了这些新的酶如何产生地球上发现的最大和最多样化的天然产物家族。重要的是，许多萜类化合物表现出有用的药用特性，例如，作为抗菌剂、抗真菌剂、抗炎剂或抗癌剂。因此，理解和工程化萜类化合物环化酶在产生具有高度特异性和效率的复杂碳支架中的功能将最终使合成化学和合成生物学的界面上的药物发现成为可能。