Nitrogen is an essential nutrient for plant growth. In the Rhizobium-legume symbiosis, root nodules are the sites of bacterial nitrogen fixation, in which atmospheric
nitrogen is converted into a form that plants can utilize. While recent studies suggested an important role for the soybean (Glycine max)
ecto-apyrase GS52 in rhizobial root hair
infection and root nodule formation, precisely how this
protein impacts the nodulation process remains undetermined. In this study, the biochemical characteristics of the GS52
enzyme were investigated. Computer modeling of the GS52
apyrase structure identified key
amino acid residues important for catalytic activity, which were subsequently mutagenized. Although the GS52
enzyme exhibited broad substrate specificity, its activity on
pyrimidine nucleotides and
diphosphate nucleotides was significantly higher than on
ATP. This result was corroborated by structural modeling of GS52, which predicted a low specificity for the
adenine base within the substrate-binding pocket of the
enzyme. The wild-type
enzyme and its inactive mutant forms were expressed in soybean roots in order to evaluate the importance of GS52 enzymatic activity for nodulation. The results indicated a clear correlation between GS52 enzymatic activity and nodule number. Altogether, our study indicates that the catalytic activity of the GS52
apyrase, likely acting on extracellular
nucleotides, is critical for rhizobial
infection and nodulation.