Mechanistic Insights of a Non-specific Inosine-Uridine Nucleoside Hydrolase (IU-NH) found in Arabidopsis thaliana

Abstract

Herbicides are crucial tools used to control unwanted vegetation to increase crop yields and landscape aesthetics. Current herbicides target metabolic pathways for protein production in plants using chemical inhibitors to inhibit metabolic pathways involved in protein synthesis, but an alternative strategy may involve targeting nucleoside salvage pathways in plants. Salvage pathways in plants recycle nucleosides to create the nucleotide monomer pools they need to synthesize DNA and RNA for protein production. The process by which nucleosides are produced varies from organism to organism, but nucleoside hydrolases are enzymes utilized by plants to catalyze purine and pyrimidine hydrolysis to create an energy efficient pathway allowing recycling of nucleobases in early seedling development. Salvage processes with nucleoside hydrolases have been found to occur in many organisms such as plants, yeast, bacteria, and protozoa, but are absent in humans. This research was conducted to understand the mechanism by which a non-specific inosine-uridine nucleoside hydrolase (IU-NH), designated URH1 from Arabidopsis thaliana catalyzes the hydrolysis of selected nucleosides. URH1 was cloned, overexpressed, and purified to greater than 95% homogeneity to determine the activity, optimum pH, oligomerization state, calcium content, and substrate specificity. In silico structural models and molecular dynamic simulations were performed with URH1 and several different nucleoside hydrolases from protozoa and plant models to determine if conserved residues were present in the active site and if any flexible loops were present. URH1 was discovered to be a non-specific nucleoside hydrolase with a broad pH range and a high activity for uridine at high concentrations. Several uninvestigated residues and flexible loops were discovered around the active site which may serve as key sites for mechanistic control.