Molecular mechanisms of nitrate reduction, NO synthesis and post-translational regulation of plant nitrate reductases

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Plant nitrate reductase (NR) is an essential homodimeric enzyme with three prosthetic groups (molybdenum cofactor, cytochrome b, FAD). Besides its primary metabolic function to reduce nitrate to nitrite, which is considered as first and rate-limiting step in plant nitrogen assimilation, NR is also able to reduce nitrite to the signaling molecule nitric monoxide (NO). Although many downstream functions and processes of NO are known, the enzymatic mechanism how NO is produced by NR remains largely unknown. During catalysis NR undergoes major conformational changes involving domain movements for efficient internal electron transfer. On protein level, 14-3-3 protein binding to phosphorylated NR via one well-known high- and one recently discovered low-affinity site regulates the enzyme activity by disturbing the internal electron transfer. It has not yet been investigated whether and how the NO synthetic activity of NR is also subject to post-translational regulation. Our goal is to understand the molecular mechanism of nitrite reduction by plant nitrate reductase as well as the interplay between nitrate and nitrite reducing activities. As model proteins, we will focus on the characterization of the two NR isoforms from Arabidopsis thaliana, AtNIA1 and AtNIA2, because it is generally assumed that AtNIA1 is mainly involved in nitrite reduction while AtNIA2 exerts predominantly nitrate reductase activity. We will recombinantly express and purify both enzymes as full-length proteins as well as functionally active domain fragments in their wildtype form and as mutants where functionally important residues have been altered. We will determine the kinetic properties of the pure enzymes for either reaction and identify crucial residues for either reaction. Furthermore, we will analyze the impact of 14-3-3s on either activity for AtNIA1 and AtNIA2 with respect to function and binding properties via activity and binding assays, in order to reveal the differences between both isoforms. As the enzymes are known to undergo conformational changes during catalysis, understanding of the structure-function relationship of NR is of great interest. Therefore, we will crystallize plant NR in its active and/or in the 14-3-3-inhibited state. Stabilization of the inhibitory complex will be required for crystallization and will be achieved by specific covalent crosslinking between the interaction partners. As prerequisite, we will identify and characterize the recently described low-affinity binding site between NR and 14-3-3, as the interacting site in 14-3-3 is yet unknown.This project aims to elucidate the mechanistic basis of nitrite reduction by NR, to reveal the distinct functions of different NR isoforms, and to understand the structural properties of NR in its active and 14-3-3-inhibited state in order to understand the dual enzyme function and the presence of dual NR isoforms in one organism.