Biological nitrogen fixation (BNF) allows plants to utilize atmospheric nitrogen for their nutrient needs, through a symbiotic relationship with rhizobia and has attracted considerable attention of the scientific community due to its potential to enhance sustainability in agriculture. Recent studies, focusing on model legume Medicago truncatula, imply an active role of glycoprotein GBP1 during nodulation, which is an important step in the establishment of the legume-bacteria relationship. The physiological properties of this protein remain poorly studied. Structural data indicate the existence of nine N-glycosylation sites plus one predicted site that could not be observed in the crystal structure. To investigate the importance of glycosylation in the physiological function of the protein, constructs with their glycosylation sites mutated were expressed in Nicotiana benthamiana and their structural stability and enzymatic activity were examined using nano differential scanning fluorimetry and reducing sugar assays.
Mutations affecting glycosylation resulted in a decrease in both protein stability and enzymatic activity, with the extent of these effects being highly site-dependent. This decreases across the mutants suggests that glycosylation contributes to both the structural integrity and functional efficiency of MtGBP1. Interestingly, the fully deglycosylated mutant apoGBP1 retained enzymatic activity and exhibited higher melting temperatures than variants with only single glycosylation site disrupted, shedding light in the non-additive effects of accumulation of mutations. Overall, these findings underline the importance of site-specific N-glycosylation in maintaining MtGBP1 stability and function and provide new insights into the role of site-specific glycosylation in regulatory role of glycosylation in enzyme activity.