Structure and Function of Biosynthetic Enzymes

生物合成酶的结构和功能

• 批准号: 8753490
• 负责人: DAVID W CHRISTIANSON
• 金额: $ 32.71万
• 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 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8753490
• 关键词: Acids; Actinic keratosis; Active Sites; Anabolism; Anti-Bacterial Agents; Anti-Inflammatory Agents; Anti-inflammatory; Antifungal Agents; Antimalarials; Antineoplastic Agents; Architecture; Artemisinins; Binding; Biological; Biological Factors; Biology; Canes; Carbon; Catalysis; Cations; Characteristics; Chemicals; Code; Commit; Complex; Cyclization; Data; Diterpenes; Engineering; Ensure; Enzymatic Biochemistry; Enzymes; Evolution; Exhibits; Family; Funding; Generations; Investigation; Kinetics; Knowledge; Learning; Libraries; Life; Malignant Neoplasms; Metals; Molecular; Natural Products Chemistry; Nature; Paclitaxel; Pathway interactions; Pharmaceutical Preparations; Plants; Premalignant; Property; Protons; Reaction; Research; Roentgen Rays; Sesquiterpenes; Site; Streptomyces coelicolor; Structure; Structure-Activity Relationship; System; Taxane Compound; Taxus; Terpenes; Terpenoid Biosynthesis Pathway; Water; Work; analog; aristolochene synthase; artemisinine; base; chemical reaction; cost effective; design; drug development; drug discovery; innovation; isoprenoid; monoterpene cyclase; mutant; nanoscale; novel; premature; prevent; public health relevance; scaffold; structural biology; synthetic biology; taxadiene synthase; taxane; terpene synthase; trichodiene synthase

DESCRIPTION (provided by applicant): The proposed research explores the structure and mechanism of terpenoid cyclases, which are unique among enzymes in that they catalyze the most complex carbon-carbon bond forming reactions in biology: on average, two-thirds of the substrate carbon atoms undergo change in bonding and/or hybridization during the course of a typical cyclization cascade to generate one or more products containing multiple rings and stereocenters. Given the vast chemodiversity of terpenoid natural products, it is notable that many terpenoids exhibit useful pharmacological properties. For example, the taxane diterpene paclitaxel (Taxol) is a blockbuster cancer chemotherapeutic drug, the sesquiterpene artemisinin is an antimalarial drug, and the diterpene ingenol (Picato) is used to treat precancerous actinic keratosis. Thus, understanding terpenoid cyclase function in generating complex carbon scaffolds enables drug discovery at the interface of natural products chemistry, enzymology, structural biology, and synthetic biology. To advance our understanding of structure-function relationships in terpenoid cyclases, and to facilitate innovative approaches for the generation of biologically active terpenoids, we will pursue the following lines of investigation: (1) we will determine how alpha-beta-gamma domain architecture influences catalysis by a diterpene cyclase, using taxadiene synthase as our paradigm. This enzyme catalyzes the first committed step of Taxol biosynthesis in the Pacific yew and has been utilized in synthetic biology approaches. (2) We will determine X-ray crystal structures of epi-isozizaene synthase and its site-specific mutants that generate alternative cyclization products to decipher the three-dimensional code that directs the sesquiterpene cyclization cascade. Ultimately, these studies will allow us to engineer enzymes that generate novel cyclic terpenoid products by design. (3) We will determine the structural and chemical basis for water management strategies in the active sites of terpenoid cyclases that utilize a reactive water molecule to quench the cyclization cascade, namely, germacradien-4-ol synthase and methylisoborneol synthase. We will also learn how the active site of aristolochene synthase, which contains an unreactive water molecule, ensures that this water molecule remains an "innocent bystander" in catalysis.

描述（由申请人提供）：拟议的研究探讨了萜类化合酶的结构和机制，它们在酶中是独一无二的，因为它们会催化生物学中最复杂的碳碳键反应：平均而言，在典型的case cascc cascccy cascc casc casc casccy casse cass cass的构造中的三分之一平均是三分之二立体中心。鉴于萜类天然产物的巨大化学多样性，值得注意的是，许多萜类化合物具有有用的药理特性。例如，紫杉烷二萜紫杉醇（紫杉醇）是一种重磅癌的化学治疗药物，倍半萜烯氨基氨基甲氨氨酸是一种抗疟药，而二培酸内酚（picato）用于治疗前癌前活性活化的活性角化作用。因此，理解萜类化合酶在产生复杂的碳支架中的功能可以在天然产物化学，酶学，结构生物学和合成生物学的界面上发现药物。 To advance our understanding of structure-function relationships in terpenoid cyclases, and to facilitate innovative approaches for the generation of biologically active terpenoids, we will pursue the following lines of investigation: (1) we will determine how alpha-beta-gamma domain architecture influences catalysis by a diterpene cyclase, using taxadiene synthase as our paradigm.这种酶催化了太平洋紫杉中紫杉醇生物合成的第一步，并已用于合成生物学方法。 （2）我们将确定Epi-Isozizaene合酶的X射线晶体结构及其位点特异性突变体，这些突变体生成替代环化产物，以破译指导倍半萜环化级联的三维代码。最终，这些研究将使我们能够设计通过设计生成新型环萜产品的酶。 （3）我们将确定使用反应性水分子淬灭环化的萜类化合酶的活性位点中水管理策略的结构和化学基础 级联，即Germacradien-4-ol合成酶和甲基异位醇合酶。我们还将学习含有不反应性水分子的阿里斯洛胆料合酶的活性位点如何确保该水分子在催化中仍然是“无辜的旁观者”。