Enzymes of the Vinca Alkaloids

长春花生物碱的酶

• 批准号: 7676297
• 负责人: Aimee Usera
• 金额: $ 4.72万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-09 至 2012-03-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7676297
• 关键词: Alkaloids; Alkynes; Amino Acid Sequence; Azides; Biochemistry; Biological; Biological Factors; Biotin; Catharanthus roseus; Cell Extracts; Cells; Chemicals; Chemistry; Complex; Crude Extracts; Detection; Development; Diagnostic radiologic examination; Diazomethane; Disease; Electrophoresis; Enzymes; Equilibrium; Gel; Indole Alkaloids; Ketones; Knowledge; Label; Lead; Methods; Modification; Molecular; Monoterpenes; Nature; Oximes; Pathway interactions; Peptide Sequence Determination; Plants; Production; Proteins; Radiolabeled; Reaction; Research; Scheme; Series; Solutions; Structure; Terpenes; Tryptamines; Vinblastine; Vinca Alkaloids; Vincristine; Western Blotting; Yohimbine; analog; base; cDNA Library; chemotherapy; cost; crosslink; effective therapy; experience; interest; member; protein crosslink; public health relevance; radiotracer; secologanin; single molecule; strictosidine; tool; tryptamine analog

DESCRIPTION (provided by applicant): Catharanthus roseus produces a wealth of biologically active alkaloids including the potent anticancer molecules vincristine and vinblastine and the antihypertension agent ajmalicine. The complex nature of the C. roseus biosynthetic pathway leads to a wide variety of valuable compounds, however, the diversity of the pathway also results in small quantities of the desired materials. A solution to the impractical amounts of medicinal compounds produced by Catharanthus roseus exists in the elucidation of the largely unknown biosynthetic pathway. The alkaloids of interest all derive from the common precursor strictosidine which is deglycosylated to form several intermediates existing in equilibrium. These-intermediates lead to one of four pathways which produce the biologically active alkaloids. This proposal focuses on the enzymes of the three less familiar pathways leading to the alkaloids ajmalicine, isositsirikine, and yohimbine. The information I obtain will ultimately aid in the reengineering of the pathway to suppress production of less useful alkaloids, thereby increasing formation of more valuable alkaloids such as the chemotherapeutics vinblastine and vincristine. I will isolate and identify at least one enzyme which catalyzes one of several of the major channels of the C. roseus biosynthetic pathway using two aims. Aim one will focus on the synthetic modification of strictosidine and tryptamine to incorporate a series of labels, for crosslinking enzymes, and tags, for detecting, into a single molecule. The labels will range from photolabile aryl azides to diazirines. The tags will include radiolabels as well as alkynes and ketones, capable of incorporating biotin labels through click chemistry and oxime formation, respectively. I will form the molecules through a combination of synthetic and chemoenzymatic reactions. The strictosidine analogs will then be assessed for enzymatic recognition in cell free extracts. The labeled and tagged analogs that are recognized by the C. roseus enzymes will be crosslinked to the proteins in cell extracts. Following isolation by 2-D electrophoresis, I will detect the labeled enzymes by radiography, in the case of radiolabeled tags, and western blot analysis when dealing with biotin tagged molecules. The Danforth Plant Center will sequence the proteins and experienced members of the lab will compare these sequences to a cDNA library. PUBLIC HEALTH RELEVANCE: The knowledge gained during my research will allow for reengineering of the pathways of C. roseus to increase production of medicinal compounds and facilitate access to practical quantities of these therapies. This information could ultimately lead to more effective and more affordable treatments of disease.

描述（由申请人提供）：长春花产生丰富的生物活性生物碱，包括有效的抗癌分子长春新碱和长春花碱以及抗高血压剂阿马利辛。 C.roseus 生物合成途径的复杂性导致产生多种有价值的化合物，然而，途径的多样性也导致所需材料的数量较少。解决长春花产生的药用化合物数量不切实际的问题在于阐明很大程度上未知的生物合成途径。感兴趣的生物碱均源自共同的前体胡豆素，其被去糖基化形成几种平衡存在的中间体。这些中间体导致产生生物活性生物碱的四种途径之一。该提案重点关注产生生物碱 ajmalicine、isosirikine 和 yohimbine 的三种不太熟悉的途径的酶。我获得的信息最终将有助于重新设计抑制不太有用的生物碱产生的途径，从而增加更有价值的生物碱的形成，例如化疗药物长春花碱和长春新碱。我将使用两个目标分离并鉴定至少一种酶，该酶催化 C.roseus 生物合成途径的几个主要通道之一。目标之一将集中于胡胡豆素和色胺的合成修饰，将一系列用于交联酶的标记和用于检测的标签合并到单个分子中。这些标签的范围从对光不稳定的芳基叠氮化物到二氮丙啶。这些标签将包括放射性标记以及炔烃和酮，能够分别通过点击化学和肟形成结合生物素标记。我将通过合成和化学酶反应的结合来形成分子。然后将评估胡胡豆苷类似物在无细胞提取物中的酶促识别。被 C.roseus 酶识别的标记和标签类似物将与细胞提取物中的蛋白质交联。通过二维电泳分离后，如果是放射性标记的标签，我将通过放射线照相技术检测标记的酶，而在处理生物素标记的分子时，我将通过蛋白质印迹分析来检测标记的酶。丹福斯植物中心将对蛋白质进行测序，经验丰富的实验室成员会将这些序列与 cDNA 文库进行比较。公共健康相关性：我在研究过程中获得的知识将允许对玫瑰花的途径进行重新设计，以增加药物化合物的产量并促进获得这些疗法的实际数量。这些信息最终可能会带来更有效、更实惠的疾病治疗方法。