Molecular, structural and functional characterization of flavonol synthase gene and its encoded protein in Millettia...

Millettia pinnata (M. pinnata) is a medicinally important plant containing many phytoconstituents capable of curing various diseases. Flavonol synthase (FLS, EC 1.14.20.6) has a significant role in flavonoid biosynthesis like kaempferol having various pharmacological properties. The present study aimed to characterize the FLS gene and its encoded protein (MpFLS) in M. pinnata comprehensively. FLS gene was characterized by isolating RNA from M. pinnata leaves, cDNA synthesis, PCR-based amplification of the desired FLS gene, sequencing of the PCR amplified product and sequence analysis followed by detailed protein-protein interaction and protein flexibility analysis. PCR product sequencing and analysis revealed that the full-length cDNA of M. pinnata FLS contains a 952-base pair long open reading frame (ORF) encoding 234 amino acids that has been submitted to NCBI (OM469017). The NCBI blastp analysis of 234 amino acid sequences of FLS shows 81.9% similarity to Vigna unguilata, whereas blastn analysis exhibited 83.31% identity to Clitoria ternatea L. that belongs to the 2OG-FeII Oxy superfamily. In silico characterization of primary, secondary, and tertiary structure, docking of MpFLS with dihydrokaempferol showed a strong binding (-10.0 kcal mol⁻¹) stabilized by hydrogen bonds (Gly54, Glu256) and hydrophobic/π-π interactions, highlighting a specific and stable enzyme-substrate complex. STRING based protein-protein interaction analysis and detailed ProtScale and MEDUSA based protein flexibility analysis of FLS protein revealed that FLS it is highly stable and hydrophilic. M. pinnata FLS gene have 952-base pair long ORF while encoding FLS protein of 234 amino acids long. FLS belong to 2-oxoglutarate/Fe(II)-dependent oxygenase superfamily that utilize molecular oxygen and 2-oxoglutarate to oxidize various substrates. Molecular docking results indicate that MpFLS binds dihydrokaempferol with high specificity and stability, supporting its key role in kaempferol biosynthesis in Pongamia pinnata. Present study provides systematic and comprehensive analysis on FLS gene and its encoded FLS protein in M. pinnata revealing that FLS protein is highly stable and hydrophilic in nature.