Molecular characterization, modeling, and docking analysis of late phytic acid biosynthesis pathway gene, inositol polyphosphate 6-/3-/5-kinase, a potential candidate for developing low phytate crops.

The coding sequence of inositol polyphosphate 6-/3-/5-kinase (GmIPK2) gene was identified and cloned from popular Indian soybean cultivar Pusa-16. The clone was predicted to encode 279 amino acids long, 30.97 kDa protein. Multiple sequence alignment revealed an inositol phosphate-binding motif, PxxxDxKxG throughout the IPK2 sequences along with other motifs unique to inositol phosphate kinase superfamily. Eight α-helices and eight β-strands in antiparallel β-sheets arrangement were predicted in the secondary structure of GmIPK2. The temporal analysis of GmIPK2 revealed maximum expression in the seed tissues during later stages of development while spatially the transcript levels were lowest in leaf and stem tissues. Endosperm-specific cis-regulatory motifs (GCN4 and Skn_1) which support high levels of expression, as observed in the developing seeds, were detected in its promoter region. The protein structure of GmIPK2 was modeled based on the crystal structure of inositol polyphosphate multikinase from Arabidopsis thaliana (PDB:4FRF) and subsequently docked with inositol phosphate ligands (PDB: 5GUG-I3P and PDB: 4A69-I0P). Molecular dynamics (MD) simulation established the structural stability of both, modeled enzyme and ligand-bound complexes. Docking in combination with trajectory analysis for 50 ns MD run confirmed the participation of Lys105, Lys126 and Arg153 residues in the formation of a network of hydrogen bonds to stabilize the ligand-receptor interaction. Results of the present study thus provide valuable information on structural and functional aspects of GmIPK2 which shall assist in strategizing our long-term goal of achieving phytic acid reduction in soybean by genetic modification of its biosynthetic pathway to develop a nutritionally enhanced crop in the future.