Structural and Functional Analysis of the Plant Phenylpropanoid Biosynthetic Pathway

这个项目的目标是了解植物苯丙素生物合成的酶机制。本研究采用分子水平上的综合实验方法，旨在了解苯丙烷类化合物生物合成途径中酶的催化性质的结构和功能基础。该项目的长期目标是使用来自蛋白质X射线结晶学的原子分辨酶结构作为起点，合理地操纵生物合成途径中单个生物合成酶和多个酶的底物和产物的特异性。这项工作集中在紫花苜蓿生产苯丙烷的一系列分支途径上。特别是，这项提议的重点是完成参与结瘤诱导剂和植物保卫素生物合成的几种酶的三维X射线晶体结构。此外，还将对结构表征的酶进行功能实验。后面这些实验的重点将是揭示控制每种酶的底物和产品专一性的核心机制原理。目前的目标是(1)扩大植物聚酮合酶、查尔酮合酶、二苯乙烯合酶和吡喃酮合酶的结构和功能分析；(3)解决查尔酮异构酶的X射线晶体结构，然后进行额外的功能实验来调节这些酶的特异性。这些研究将阐明在植物中控制苯丙烷产生的酶机制。此外，这一实验策略使用了蛋白质X射线结晶学、单一和多个氨基酸位置的定点和饱和突变、从生物合成酶同源家族收集的序列信息、小分子鉴定和动力学分析，将作为合理操作底物和生物合成途径中单个生物合成酶和多个酶的产物特异性的起点。植物苯丙烷类化合物是植物在受到真菌、细菌和病毒等侵染物侵袭时合成的一组不同的化学物质。这些复杂的小分子在植物与周围环境的相互作用中也发挥着至关重要的作用。这类天然产物包括水杨酸酯、木脂素、绿原酸、香豆素、二苯乙烯和类黄酮类。类黄酮族包括异黄酮类、胡萝卜素、黄酮类、黄酮醇和花青素。这种分子多样性是由于被称为酶的高度特异的生物催化剂的作用而产生的。这些化合物在植物中的用途多种多样，包括作为结构聚合物、防御屏障、响应微生物、昆虫和食草动物捕食而合成的防御化学物质、固氮根瘤菌的信号分子、紫外线保护剂和色素。除了在植物生理学中的作用外，苯丙烷类化合物还具有许多已被证明对制药、食品、农业和营养业有用的特性。特别是，一些植物来源的苯丙烷类化合物是有价值的药剂。此外，经常食用苯丙烷类化合物，包括木脂素、二苯乙烯类和异黄酮类化合物，对健康有相当大的好处。该项目利用一种名为蛋白质x射线结晶学的技术来破译重要植物催化剂的三维形状，这些催化剂负责产生苯丙烷小分子。然后，这些信息被用来理解和改变这些催化剂的特异性，以便在这些生物合成系统中创造更大的分子多样性。这些酶的底物和产物特异性的调节将直接影响生产具有治疗和农业意义的新化合物的努力。

The goal of this project is to understand the enzymatic mechanisms underlying the biosynthesis of plant phenylpropanoids. This research employs a comprehensive experimental approach on the molecular level aimed at understanding the structural and functional basis for the catalytic properties of enzymes involved in phenylpropanoid biosynthetic pathways. The long-term objective of this project is to use atomic resolution enzyme structures derived from protein x-ray crystallography as starting points for the rational manipulation of both the substrate and product specificity of individual biosynthetic enzymes and multiple enzymes in biosynthetic pathways. The work centers on a branched set of pathways for phenylpropanoid production in alfalfa. In particular, this proposal focuses on the completion of the three-dimensional x-ray crystal structures of several enzymes involved in the biosynthesis of nodulation inducers and phytoalexins. In addition, functional experiments on the structurally characterized enzymes will be carried out. The emphasis of these latter experiments will be to uncover the core mechanistic principles governing the substrate and product specificity of each enzyme. The current objectives are (1) to expand the structural and functional analysis of the plant polyketide synthases, chalcone synthase, (2) stilbene synthase, and pyrone synthase; and (3) to solve the x-ray crystallographic structure of the enzyme chalcone isomerase followed by additional functional experiments to modulate the specificity of these enzymes. These studies will elucidate the enzymatic mechanisms governing phenylpropanoid production in plants. In addition, this experimental strategy employing protein x-ray crystallography, site-directed and saturation mutagenesis of single and multiple amino acid positions, sequence information gathered from homologous families of biosynthetic enzymes, small molecule identification, and kinetic analyses will serve as starting points for the rational manipulation of the substrate and product specificity of individual biosynthetic enzymes and multiple enzymes in biosynthetic pathways. Plant phenylpropanoids are a diverse group of chemicals synthesized by plants when they are challenged with infectious agents such as fungi, bacteria, and viruses. These complex small molecules also play vital roles in the interaction of plants with their surrounding environment. This family of natural products includes salicylates, lignans, chlorogenic acid, coumarins, stilbenes, and flavonoids. The flavonoid group encompasses isoflavones, pterocarpans, flavones, flavonols, and anthocyanins. This molecular diversity arises due to the action of highly specific biological catalysts known as enzymes. The utility of these compounds in plants varies widely and includes roles as structural polymers, defense barriers, defense chemicals synthesized in response to microbial, insect, and herbivore predation, signaling molecules for nitrogen-fixing rhizobia bacteria, UV-protective agents, and pigments. In addition to their role in plant physiology, phenylpropanoids possess a number of properties that have proven useful to the pharmaceutical, food, agricultural, and nutritional industries. In particular, a number of plant-derived phenylpropanoids are valuable medicinal agents. Moreover, the regular dietary consumption of phenylpropanoid-derived compounds including lignans, stilbenes, and isoflavonoids has considerable health benefits. This project utilizes a technique known as protein x-ray crystallography to decipher the three dimensional shapes of the vital plant catalysts responsible for creating phenylpropanoid small molecules. This information is then used to understand and alter the specificity of these catalysts in order to create even greater molecular diversity in these biosynthetic systems. Modulation of the substrate and product specificity of these enzymes will directly impact efforts to produce novel compounds of both therapeutic and agricultural interest.