Structural and Functional Analysis of the Plant Phenylpropanoid Biosynthetic Pathway

The Salk Institute For Biological Studies, La Jolla CA

Investigators

Abstract

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.