Enzymes of the Vinca Alkaloids

描述(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，以及降压药阿马利辛。玫瑰花生菌生物合成途径的复杂性导致了大量有价值的化合物的产生，然而，该途径的多样性也导致了所需材料的少量。长春花产生的药用化合物的不切实际数量的解决方案存在于对基本上未知的生物合成途径的阐明中。感兴趣的生物碱都来自共同的前体斯特拉托苷，它被脱糖形成几个平衡存在的中间体。这些中间体通向产生生物活性生物碱的四条途径之一。这项提案侧重于三种不太为人所知的途径的酶，这三种途径通向生物碱阿玛利辛、异山梨碱和育亨宾。我获得的信息最终将有助于途径的重组，以抑制不太有用的生物碱的产生，从而增加更有价值的生物碱的形成，如化疗药物长春花碱和长春新碱。我将使用两个AIMS分离和鉴定至少一种催化玫瑰红曲霉生物合成途径的几个主要通道之一的酶。目的一将重点放在合成修饰上，以将一系列用于交联酶的标记和用于检测的标记结合到单个分子中。这些标签的范围从不发光的芳基叠氮化合物到二叠氮类化合物。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酮类形成结合生物素标记。我将通过合成酶反应和化学酶反应的组合来形成分子。然后，将评估这些严格的皂苷类似物在无细胞提取物中的酶识别能力。被玫瑰红曲霉酶识别的标记和标记的类似物将与细胞提取液中的蛋白质交联。在通过二维电泳法分离后，我将通过放射照相来检测标记的酶，对于放射性标记的标记，我将通过蛋白质印迹分析来检测标记的生物素分子。丹福斯植物中心将对这些蛋白质进行测序，经验丰富的实验室成员将把这些序列与一个cdna文库进行比较。与公共卫生相关：在我的研究期间获得的知识将允许对玫瑰弧菌的途径进行重新设计，以增加药物化合物的生产，并促进获得实际数量的这些疗法。这些信息最终可能导致更有效和更负担得起的疾病治疗。

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.