In-silico Analysis and Structural Modelling of Peroxidase Enzyme in Soybean [Glycine max (L.) Merrill]

Abstract

Aims: Seed deterioration is a vital constraint in soybean seeds and peroxidase enzyme plays an important role in seed viability during storage. The study aimed to characterise the soybean (Glycine max [L.] Merr.) seed-coat peroxidase (Ep) gene through in silico approaches and to elucidate its structural and evolutionary features related to oxidative-stress tolerance and seed storability. Study Design: A computational experimental design was followed, integrating sequence alignment, structural prediction, and phylogenetic analysis of Ep and related Glycine max peroxidase isoforms. Place and Duration of Study: The bioinformatics analyses were conducted at the Department of Seed Science and Technology, College of Agriculture, University of Agricultural Sciences, Bangalore, between April 2025 and June 2025. Methodology: The full-length Ep peroxidase sequence (GenBank accession no. L78163.1) was retrieved from NCBI and analysed using ClustalW in BioEdit v7.2.5 for multiple-sequence alignment. Structural prediction and secondary-structure composition were obtained using trRosetta, and conserved catalytic residues were identified. The aligned dataset was used for phylogenetic tree construction in MEGA X (v11) employing the Neighbour-Joining method with 1000 bootstrap replications, using the Actin gene (J01298.1) as an outgroup. Results: The Ep protein exhibited a typical class III peroxidase fold, with approximately 42 % α-helices, 15 % β-strands, and 43 % random coils and loops. The catalytic triad (His-42, Arg-38, His-170) and disulfide-forming cysteine residues were conserved across all peroxidase isoforms. Phylogenetic analysis resolved three major clades with bootstrap support of 72–100 %, confirming evolutionary stability and close homology among soybean peroxidases. Conclusion: The in silico analysis demonstrated that the Ep peroxidase gene retains a conserved catalytic geometry and robust α-helical framework, contributing to oxidative-stress tolerance in soybean seeds. These findings provide a molecular basis for the role of peroxidase in seed longevity and offer potential markers for breeding programmes aimed at improving seed storability.