Characterization of the active site of a germin like protein 1 as an oxidative stress defense enzyme in plants

Glycosylated proteins like germin-like proteins (GLPs) are incredibly diverse inside the kingdom Plantae, and mostly GLPs exhibit superoxide dismutase (SOD) function. Identification of catalytic residues is important for understanding the mechanism of enzyme-catalyzed reactions. The increased bioactivity of SOD was observed when OsRGLP1 was over-expressed in tobacco. The purpose of the current work was to identify and characterize the active site of OsRGLP1. Bioinformatics tools were used to predict the three-dimensional structure of OsRGLP1 and the shape of residues implicated in the substrate and metal ion binding. The role of predicted active site residues (E116, H109, H111, and H157) in the structure-function relationship in OsRGLP1 was investigated by site-directed mutagenesis where each residue was substituted with glycine. These amino acids are highly conserved among GLP family and structural data have implicated these residues in substrate binding at the active site. Transient transformation of tobacco plants was performed to further study these loss-of-function mutants. To investigate the impact of the mutation on SOD activity, these transgenic plants were employed as a source of mutant and native proteins for SOD activity assays. The SOD assay results revealed a complete loss of activity in all mutants, supporting the crucial role of these residues for metal ion binding in the enzyme active site.