[P001] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P01-031] Identification of the catalytic residues and reaction mechanism of arginine synthetase

○Yukihiro Okamoto¹, Yuta Michimori¹, Haruyuki Atomi¹ (1. Kyoto Univ. (Japan))

Keywords:

archaea,arginine synthetase,reaction mechanism

A novel enzyme involved in arginine catabolism was discovered in the hyperthermophilic archaeon Thermococcus kodakarensis. The enzyme, designated arginine synthetase, catalyzes the conversion of arginine into citrulline and ammonia coupled to the production of ATP from ADP and Pi. While the enzyme catalyzes the same deimination reaction of arginine to citrulline as the conventional arginine deiminase, it is unique in that, unlike arginine deiminase which catalyzes irreversible hydrolysis, it can catalyze an energy-conserving, reversible reaction. Arginine synthetase differs from arginine deiminase in both its primary and tertiary structures, and belongs to a completely different protein family. Moreover, arginine synthetase shows little similarity to previously studied proteins, both structurally and in terms of the catalytic reaction. There is thus no knowledge about its catalytic residues or reaction mechanism. This study aimed to identify the catalytic residues of arginine synthetase from T. kodakarensis, and to elucidate the mechanism of catalysis. In order to predict the catalytic residues, we prepared multiple sequence alignments of arginine synthetase homologs, and searched for well-conserved residues. In addition, the active site region was predicted by docking simulations with the substrate using Alphafold2/3 and AutoDock Vina, and residues thought to be involved in the reaction were selected. Position-specific substitutions were introduced at each residue, and individual genes were expressed in Escherichia coli. After the recombinant proteins were purified, activity in reaction mixtures with varying substrate concentrations was measured to investigate the effects of residue exchange on substrate recognition. To analyze the reaction mechanism, substrates containing isotopic elements were added to the reaction solution. By identifying the types of isotopes incorporated into the product, the fate of each atom in the substrate was traced, enabling us to propose a reaction mechanism for arginine synthetase.