Enzymatic Cyclization to Labdanes and Related Diterpenoid Natural Products

酶促环化生成拉丹烷类及相关二萜类天然产物

• 批准号: 7476305
• 负责人: REUBEN JOHN PETERS
• 金额: $ 27万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7476305
• 关键词: Acids; Active Sites; Alkenes; Anabolism; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Antimicrobial Effect; Applications Grants; Area; Biochemistry; Biological; Biological Factors; C-terminal; Catalysis; Chemical Structure; Chemicals; Class; Classification; Cloning; Collaborations; Complex; Coupled; Crystallography; Cyclization; DNA Sequence Rearrangement; Diphosphates; Diterpenes; Drug Industry; Elements; Engineering; Environment; Enzymatic Biochemistry; Enzymes; Escherichia coli; Exhibits; Family; Figs - dietary; Foundations; Future; Goals; Heterogeneity; Hydrocarbons; Illinois; Individual; Investigation; Isomerism; Knowledge; Labdanes; Libraries; Mediating; Medical; Metabolic; Metabolic Pathway; Metabolism; Minor; Modeling; Modification; Molecular Conformation; N-terminal; Numbers; Outcome; Pathway interactions; Pennsylvania; Pharmacologic Substance; Phytochemical; Plants; Principal Investigator; Production; Publishing; Purpose; Range; Reaction; Refractory; Relative (related person); Research; Research Personnel; Site; Site-Directed Mutagenesis; Skeletal system; Source; Specific qualifier value; Specificity; Structure; Structure-Activity Relationship; Substrate Specificity; System; Terpenes; Testing; Triterpenes; Vertebral column; Work; analog; anticancer activity; base; combinatorial; copalyl diphosphate; ent-kaurene synthetase A; experience; geranylgeranyl diphosphate; improved; innovation; insight; interest; ionization; isoprenoid; labdane; member; novel; pimara-7,15-diene; prenyl; programs; protonation; terpene synthase

DESCRIPTION (provided by applicant): Labdanes and related diterpenoids form a large group of almost 7,000 organic compounds that display a wide range of biological activity and includes a number with significant pharmaceutical activity, particularly medically relevant anticancer, anti-inflammatory, or antimicrobial effects. In addition, potential uses for many of these generally scarce natural products have yet to be fully explored. Biosynthesis of these complex compounds is initiated via a poorly understood acid/base catalyzed (class II) reaction, which forms the bicyclic core structure that defines this super-family of natural products. This bicyclic core is then specifically elaborated by a more typical allylic diphosphate ionization driven (class I) reaction. Interestingly, these mechanistically distinct reactions are mediated by phylogenetically related terpene synthases, albeit in different active sites. Despite the functional importance of the two classes of labdane-related diterpene synthases in initiating the biosynthesis of large numbers of natural products with both realized and potential medical significance, little is known about the enzymatic determinants underlying substrate and product specificity. We propose to investigate these consecutive cyclization reactions as a first step towards our long-term goal of engineering terpenoid natural product biosynthesis for pharmaceutical purposes. On the basis of our previously published and preliminary studies, we hypothesize that the class I active site resides in the C-terminal domain, while the class II active site lies at the interface between two other domains. The structurally defined C-terminal domain associated with class I reactions enables immediate study of the observed specificity via macromolecular modeling directed mutational analysis. To characterize class II cyclization more basic mechanistic enzymology studies are proposed to identify the determinants for catalysis, as well as initial investigations of product specificity. In addition, for both classes of reactions we expect to obtain significant insights through collaborative experimental structure determination, and via continued identification of functionally novel enzymes, along with initial metabolic engineering studies. Thus, the proposed studies will elucidate the structure-function relationships underlying the consecutive class I I/class I cyclization reactions in labdane-related diterpene biosynthesis, and will have a broader impact in further increasing our understanding and use of terpenoid enzymatic cyclization more generally.

描述（由申请人提供）：Labdanes和相关的二萜类化合物构成了近7000种有机化合物，具有广泛的生物活性，包括一些具有重要的药物活性，特别是与医学相关的抗癌、抗炎或抗菌作用。此外，许多这些普遍稀缺的天然产品的潜在用途尚未得到充分探索。这些复杂化合物的生物合成是通过一个鲜为人知的酸/碱催化（II类）反应开始的，该反应形成了双环核心结构，定义了这个超家族的天然产物。这个双环核心然后具体阐述了一个更典型的烯丙基二磷酸离子驱动（类）的反应。有趣的是，这些机制上不同的反应是由系统发育相关的萜烯合成酶介导的，尽管在不同的活性位点。尽管两类与labdane相关的二萜合成酶在启动大量天然产物的生物合成方面具有重要的功能，具有已实现的和潜在的医学意义，但对底物和产物特异性的酶决定因素知之甚少。我们建议研究这些连续的环化反应，作为我们实现工程萜类天然产物生物合成用于制药目的的长期目标的第一步。根据我们之前发表的和初步的研究，我们假设I类活性位点位于c端结构域，而II类活性位点位于其他两个结构域之间的界面。结构上定义的c端结构域与I类反应相关，可以通过大分子模型定向突变分析直接研究观察到的特异性。为了表征II类环化，提出了更基本的机制酶学研究，以确定催化的决定因素，以及对产物特异性的初步研究。此外，对于这两类反应，我们希望通过协作实验结构确定，通过功能新颖酶的持续鉴定，以及初步的代谢工程研究，获得重要的见解。因此，所提出的研究将阐明在labdane相关的二萜生物合成中连续的I类I/ I类环化反应的结构-功能关系，并将对进一步提高我们对萜类酶环化的理解和更广泛的使用产生更广泛的影响。