Enzymes of the Vinca Alkaloids

性状（由申请方提供）：长春花产生大量生物活性生物碱，包括强效抗癌分子长春新碱和长春碱以及抗高血压药物阿曲马林。C. roseus生物合成途径导致多种有价值的化合物，然而，该途径的多样性也导致少量的所需材料。解决长春花产生的药用化合物的不切实际的量存在于阐明在很大程度上未知的生物合成途径。感兴趣的生物碱都来源于共同的前体strictosidine，其被去糖基化以形成平衡存在的几个中间体。这些中间体导致产生生物活性生物碱的四种途径之一。该建议侧重于三个不太熟悉的途径，导致生物碱ajmalicine，isositsirikine，育亨宾的酶。我所获得的信息将最终有助于重组该途径，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，如化疗药物长春碱和长春新碱。我将分离并鉴定至少一种催化C. roseus生物合成途径使用两个目标。目的一是对异木果苷和色胺进行合成修饰，将一系列用于交联酶的标记物和用于检测的标记物整合到单个分子中。标记物的范围从对光不稳定的芳基叠氮化物到二氮杂环丙烷。标签将包括放射性标记以及炔和酮，能够分别通过点击化学和肟形成掺入生物素标记。我将通过合成和化学酶反应的结合来形成分子。然后将评估异胡荽苷类似物在无细胞提取物中的酶识别。C.玫瑰酶将与细胞提取物中的蛋白质交联。在通过2-D电泳分离之后，我将通过放射照相术检测标记的酶，在放射性标记的情况下，以及在处理生物素标记的分子时进行蛋白质印迹分析。丹佛斯植物中心将对蛋白质进行测序，实验室的经验丰富的成员将把这些序列与cDNA文库进行比较。公共卫生相关性：我在研究期间获得的知识将允许重新设计C的途径。玫瑰，以增加生产药用化合物，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.