Nitrogen is an essential nutrient in plants including many crops. The storage and remobilization of nitrogen constitutes the main metabolic process for growth and development of plants. Ureide pathway is the lately characterized metabolic route for purine degradation and is conserved in plants, as well as some bacteria and fungi. The catabolic pathway catalyzes in a stepwise manner a conversion of N-rich uric acid into glyoxylate, with the release of ammonia, and plays a pivotal role in the storage and recovery of nitrogen from metabolites. In Next Generation BioGreen21 project, we aim to understand structural and functional features of enzymes involved in this nitrogen recycling pathway, by using genes from Arabidopsis thaliana. In this study, we report our current progress on this project including two different enzymes; ureidoglycine aminohydrolase (UGlyAH), and ureidoglycolate amidohydrolase (UAH). In UGlyAH, the metal-binding site plays a crucial role in catalysis, with a release of ammonia. We were able to characterize catalytic residues in the active site and provides a detailed view of a metal-dependent enzyme mechanism. Recently, we were able to characterize structural properties of UAH. Based on our analysis, we are performing enzymatic analysis to identify functional aspects of the enzyme. Taken together, these studies would provide a novel functional feature of the enzymes involved in the nitrogen recycling pathway and could serve as a framework to develop crops with an enhanced N-efficiency.

The ureide pathway has recently been identified as the metabolic route of purine catabolism in plants and some bacteria. In this pathway, uric acid, which is a major product of the early stage of purine catabolism, is degraded into glyoxylate and ammonia via stepwise reactions of seven different enzymes. Therefore, the pathway has a possible physiological role in mobilization of purine ring nitrogen for further assimilation. (S)-Ureidoglycine aminohydrolase enzyme converts (S)-ureidoglycine into (S)-ureidoglycolate and ammonia, providing the final substrate to the pathway. Here, we report a structural and functional analysis of this enzyme from Arabidopsis thaliana (AtUGlyAH). The crystal structure of AtUGlyAH in the apo-form shows a monomer structure in the bi-cupin fold of the β-barrel and an octameric functional unit, as well as an Mn2+ ion binding site. The structure of AtUGlyAH in complex with (S)-ureidoglycine revealed that the Mn2+ ion acts as a molecular anchor to bind (S)-ureidoglycine and its binding mode dictates the enantioselectivity of the reaction. Further kinetic analysis characterized the functional roles of the active site residues, including the Mn2+ ion binding site and residues in the vicinity of (S)-ureidoglycine. These analyses provide molecular insights into the structure of the enzyme and its possible catalytic mechanism.