Organization of Isoflavonoid Biosynthetic Enzymes

异黄酮是几乎只存在于豆科植物中的酚类次生代谢物。这些化合物在许多生物和非生物应激反应中起着关键作用。普遍存在的苯丙烷途径为异黄酮的生产提供了底物。一组豆类特有的酶，包括异黄酮合成酶和II型查尔酮异构酶，作为代谢分支点，将黄酮转化为异黄酮。黄酮类化合物和异黄酮类化合物的产生受到不同环境胁迫的不同调控。在特定条件下，细胞究竟如何引导共同底物流向每条途径尚不清楚。先前的研究表明，关键的类黄酮和异黄酮生物合成酶形成一个大分子复合物或一个代谢物，将代谢物输送到该途径的分支。多种证据表明，特定的酶相互作用有时是非豆科植物异黄酮生物合成代谢工程的主要瓶颈。近年来，通过在酵母中表达关键代谢酶并在培养基中提供多种底物，已经成功地重建了部分黄酮类和异黄酮类途径。令人惊讶的是，黄酮类化合物底物在酵母中的流动受到特定酶关联的影响。因此，这个体内酵母系统为我们研究代谢物的通量和关键酶的相互作用提供了一个独特而有趣的平台。利用生物化学、计算生物学和细胞生物学的综合方法，本项目旨在研究酵母代谢对异黄酮生物合成的影响，并探索植物在生物和非生物胁迫响应过程中的差异蛋白-蛋白相互作用。了解这些假定的多酶复合物的结构和功能是非常重要的，因为许多其他需要多种酶协同作用的初级和次级代谢过程可能也采用类似的调节机制。本项目的目的是量化黄酮类化合物和异黄酮类化合物在转基因酵母中各分支通路的通量，并测量特定酶相互作用对代谢物产生的影响。利用这一独特的异源系统，该项目试图通过标准的体外和体内试验来确认观察到的蛋白质-蛋白质相互作用，并建立一个数学模型来涵盖基因表达、酶动力学和蛋白质相互作用的影响。更广泛的影响：这个研究项目的高度协作性质，跨越了生物化学、计算建模和细胞生物学，为本科生、博士后助理和访问学者提供了一个拓宽科学家视野的环境。丹佛斯中心和玛丽维尔大学的院系之间拟议的合作是独特和互补的：它为一所小型文理学院的教师和学生提供了最新的研究经验；同时，它还加强了丹佛斯中心的教育和外联部分。

Isoflavonoids are phenolic secondary metabolites found almost exclusively in legumes. These compounds play key roles in many biotic and abiotic stress responses. The ubiquitous phenylpropanoid pathway provides substrates for isoflavonoid production. A set of legumespecific enzymes, including isoflavone synthase and Type II chalcone isomerase, act as a metabolic branch-point by converting flavanones to isoflavones. The production of flavonoids and isoflavonoids are differentially regulated in response to different environmental stresses. Exactly how cells direct the flow of common substrates towards each pathway under specific conditions is not clear. Previous studies demonstrated that key flavonoid and isoflavonoid biosynthetic enzymes form a macromolecular complex or a metabolon that channels metabolites into branches of the pathway. Multiple lines of evidence support that specific enzyme interactions are sometimes the main bottlenecks for metabolic engineering of isoflavone biosynthesis in non-legume plants. Recently, part of the flavonoid and isoflavonoid pathway has been reconstituted successfully in yeast by expressing key metabolic enzymes and supplying various substrates in the culture media. Surprisingly, the flow of flavonoid substrates in yeast was influenced by specific enzyme associations. This in vivo yeast system thus provides us a unique and interesting platform for studying the flux of metabolites and interactions of key enzymes. Using integrated biochemical, computational and cell biology approaches, this project aims to examine the impact of the metabolon on isoflavonoid biosynthesis in yeast and to explore the differential protein-protein interactions during biotic and abiotic stress responses in plants. It is very important to understand the structure and function of these putative multi-enzyme complexes because many other primary and secondary metabolic processes that require concerted action of multiple enzymes might well employ similar regulation mechanisms. The objective of this project is to quantify the flux of flavonoid and isoflavonoid compounds in each branch of the pathway in transgenic yeasts, and to measure the effects of specific enzyme interactions on metabolite productions. Taking advantage of this unique heterologous system, the project seek to confirm the observed protein-protein interactions with standard in vitro and in vivo assays, and to establish a mathematical model to encompass the effects of gene expression, enzyme kinetics, and protein interactions.Broader Impacts: The highly collaborative nature of this research project, which crosses between biochemistry, computational modeling, and cell biology, offers an environment for undergraduate students, postdoctoral associates, and visiting scholars to broaden as scientists. The proposed collaboration between faculties of the Danforth Center and the Maryville University is unique and complementary: It opens the up-to-date research experience to the teachers and students of a small liberal art college; at the same time, it enhances the educational and outreach component of the Danforth Center.