Molecular Analysis of Two CC-Type Glutaredoxins from the Model Legume Lotus japonicus

Glutaredoxins (GRXs) are small oxidoreductases involved in redox regulation, but the biochemical properties of plant-specific CC-type GRXs remain poorly understood, particularly in legumes. In a previous transcriptomic analysis, two CC-type glutaredoxins from Lotus japonicus, LjGRX460 and LjGRX569, were identified as differentially expressed during symbiosis with nitrogen-fixing rhizobia. Here, both proteins were produced recombinantly and characterized by sequence analysis, structural modelling and in vitro biochemical assays. Phylogenetic and sequence studies confirmed that both proteins belong to the plant-specific CC-type GRX class but differ in active site composition and overall sequence organization. Homology modelling and molecular dynamics simulations revealed distinct conformational properties, including differences in active site accessibility, electrostatic surface distribution and putative oxidation-dependent structural rearrangements. Comparative analyses with representative class I and II GRXs supported substantial structural divergence, suggesting functional specialization. Optimized expression and purification protocols yielded soluble recombinant proteins. Both LjGRX460 and LjGRX569 displayed a strong tendency to form soluble high molecular weight aggregates, similar to the class I control GRX. Enzymatic assays showed low oxidoreductase activity towards classical disulfide substrates compared with class I GRXs, while molecular docking suggested reduced affinity for bis(2-hydroxyethyl) disulfide (HEDS) and preferential interaction with L-cystine. These results indicate that LjGRX460 and LjGRX569 are unlikely to function as classical oxidoreductases and may instead perform specialized regulatory functions.