Structural and Functional Characterization of the Peroxidase Gene in Chickpea under Aluminum Stress

Aluminum (Al3+) toxicity is a significant limitation to chickpea (Cicer arietinum L.) productivity in acidic soils by impairing root growth and increasing reactive oxygen species (ROS). Class III peroxidases are major antioxidant enzymes important for ROS scavenging; however, the structural and functional role of peroxidases in chickpea under Al³⁺ stress remains unclear. This study conducted comprehensive bioinformatics and molecular docking of the chickpea peroxidase 43 isoform X2 gene. BLASTn and BLASTx analyses confirmed that the gene encodes a peroxidase-like protein. Further comparative analysis of peroxidase 43 from nine legumes revealed a high degree of sequence conservation, especially with Medicago truncatula, Pisum sativum, and Vigna radiata. Multiple sequence alignment with phylogenetic analysis highlighted conserved motifs for the catalytic mechanism and evolutionary divergence, with Cajanus cajan having the lowest conservation. The analysis of conserved domains confirmed that peroxidases have specific motifs, while 3D structure prediction confirmed the integrity of the protein fold. Protein-protein interaction analysis revealed a strong connection to enzymes involved in oxidative stress. In addition, molecular docking results revealed that the Al³⁺ ion has a strong binding affinity to the active site, which could destabilize enzymatic activity and function. In contrast, Fe2+ supports activity and function, while Cu2+ strongly competitively binds to peroxidase. Overall, these results give insight into peroxidase-mediated antioxidant defense mechanisms as well as their potential use in breeding strategies for Al3+-tolerant chickpea varieties