Enzyme Organization and Flux Control of the Phenylpropanoid Pathway

该奖项是根据2009年美国复苏和再投资法案（公法111-15）资助的。智力优势：高等植物中的苯丙素途径产生许多自然界的彩色化合物。在这些化合物中，木质素和黄酮类化合物在植物生长发育中起着重要作用。木质素是植物次生细胞壁的主要成分。黄酮类化合物在生物和非生物应激反应中具有多种功能。木质素和类黄酮合起来约占植物生物量的30%。了解这些化合物的生物合成是生物燃料研究的重要组成部分。木质素和黄酮类化合物都是由相同的起始分子——氨基酸苯丙氨酸合成的。以往的研究表明，这两组代谢物的生物合成是紧密相连的。中断木质素的生物合成可以增加类黄酮的积累，阻断生长素的运输，并阻止拟南芥的生长。然而，目前尚不清楚植物细胞如何在竞争途径之间分配共同的底物和中间体。这个项目的重点是多酶组织的性质和通量调节。多酶复合物中的酶相互作用可以改变途径通量，成为代谢途径的重要调控因子。以前，通过建立关键生物合成酶的物理接触，成功地提高了途径效率。这些融合蛋白已被用于改造植物和重组酵母的途径。本文将研究木质素与类黄酮途径交界处的多酶复合物。科学目标如下：1)量化转基因酵母中苯丙类化合物在各通路分支中的通量，并测量特定酶相互作用对代谢物产生的影响。这种独特的异种系统将被用来证明蛋白质与标准的体外和体内的相互作用。2)研究野生型拟南芥和各种代谢突变体中酶的相互作用。在野生型和突变型背景下，用激发子或紫外线处理的植物组织中关键酶的亚细胞共定位将被检查。定义以4cl为中心的多酶复合物的潜在酶相互作用将被表征。更广泛的影响：教育和推广计划是这个项目的一个组成部分。积极上进的本科生将被录取为暑期实习生。此外，丹佛斯中心和玛丽维尔大学的教职员工之间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，这项研究将加强丹佛斯中心的教育和推广组成部分。在体内酵母表达系统将发展成为一个多功能的教学工具，本科生物学实验课程。这个跨越生物化学、微生物学和细胞生物学的研究项目具有高度协作的性质，为高中生、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。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-15).Intellectual Merit: The phenylpropanoid pathway in higher plants produces many of nature's colorful compounds. Among these compounds, lignins and flavonoids play essential roles in plant growth and development. Lignins are a major component of plant secondary cell walls. Flavonoids serve multiple functions during biotic and abiotic stress responses. Lignins and flavonoids together account for approximately 30% of plant biomass. Understanding the biosynthesis of these compounds is an important part of biofuel research. Both lignins and flavonoids are synthesized from the same starting molecule, the amino acid phenylalanine. Previous studies showed that the biosyntheses of these two groups of metabolites are tightly linked. Interruptions of lignin biosynthesis could increase flavonoid accumulations, block auxin transport, and arrest the growth of Arabidopsis. It is not understood, however, exactly how plant cells distribute the common substrate and intermediates between the competing pathways. This project is focused on the nature of multi-enzyme organizations and flux regulations. Enzyme interactions in the multi-enzyme complexes could alter pathway flux, thus becoming an important regulatory factor of metabolic pathways. Previously, pathway efficiency was successfully increased by establishing physical contacts of key biosynthetic enzymes. These fusion proteins have been used to engineer plants and reconstituted pathways in yeast. Here, the multi-enzyme complex at the junction of lignin and flavonoid pathway will be studied. The scientific objectives are as follows: 1) To quantify the flux of phenylpropanoid compounds in each branch of the pathway in transgenic yeast, and measure the effects of specific enzyme interactions on metabolite productions. This unique heterologous system will be used to prove protein-protein interactions with standard in vitro and in vivo assays. 2) To study the enzyme interactions in wild type Arabidopsis and in various metabolic mutants. The sub-cellular co-localization of key enzymes in plant tissues that are treated with elicitors or UV-light in both wild type and mutant background will be examined. The potential enzyme interactions that define the 4CL-centered multi-enzyme complex will be characterized. Broader Impacts: The educational and outreach program is an integral part of this project. Highly motivated undergraduate students will be enrolled as summer interns. In addition, the collaboration among faculty members of the Danforth Center and Maryville University is unique and complimentary. It enables an up-to-date research experience for the teachers and students of a small liberal arts college. At the same time, this research will enhance the educational and outreach component of the Danforth Center. The in vivo yeast expression system will be developed as a versatile teaching tool for undergraduate biology laboratory courses. The highly collaborative nature of this research project, which crosses between biochemistry, microbiology, and cell biology, offers an environment for high school students, undergraduate students, postdoctoral associates, and visiting scholars to broaden as scientists.