Plant uricases are evolutionarily conserved but functionally divergent: A case study of Oryza coarctata uricase

Allantoin plays a major role in nitrogen mobilization as well as abiotic stress tolerance in plants, and uricase enzyme is the key rate-limiting enzyme for the biosynthesis of allantoin in peroxisomes. However, the molecular mechanisms underlying stress-induced allantoin accumulation as well as enhanced uricase activity, along with the evolutionary conservation, structural diversification and regulatory characteristics of plant uricase enzymes, remain poorly understood. This study investigates evolutionary relationships, functional variations and catalytic mechanisms of the uricase enzymes among 157 plant species. Phylogenetic analysis grouped these species into three major clades, indicating a progression from thallophytes to higher plants. Motif analysis revealed the presences of a conserved Pfam01014 (uricase) domain across the plant species. Uricase activity analysis of 22 representative species showed the highest activity in ureidic legumes, including Vigna radiata and Glycine max, and the lowest in non-leguminous species such as Cucumis sativus. Moreover, promoter analysis of the uricase gene identified diverse cis-regulatory elements associated with stress, hormone responses and plant development. Structural modelling, docking and sequence alignments revealed conserved substrate-binding residues. Biophysical characterization of recombinantly purified uricase from Oryza coarctata using fluorescence spectroscopy demonstrated uric acid binding with a dissociation constant (K_D) of 10.89 μM, positive cooperativity and the Michaelis constant (K_M) of 23.96 μM. Circular dichroism showed a conformational shift from α-helix to β-sheet upon ligand binding and molecular dynamics simulations supported stable urate interactions in O. coarctata. This work highlights the evolutionary conservation and functional divergence of uricase, featuring unique catalytic adaptations in ureidic legumes.