In silico characterization of GbPAL, GbCHS, GbDFR and GbANS structural genes involved in the biosynthesis of flavonoids in Gynura bicolor DC

Gynura bicolor is a member of the Asteraceae family, possessing important pharmaceutical compounds such as flavonoids, anthocyanins, and terpenoids. Genetic and molecular studies focusing on the biosynthesis of flavonoids are indispensable to determining their regulation. GbPAL, GbCHS, GbDFR, and GbANS are genes encoding Phenylalanine ammonia lyase, Chalcone synthase, Dihydroflavonol reductase, and Anthocyanidin synthase enzymes, resepectively and are involved in the biosynthesis of flavonoids in G. bicolor, resepectively. There are still a few studies concerning the characterization of these genes. In this study, the structural enzyme sequences were selected to be characterized in silico using bioinformatics software. The phylogenetic analysis demonstrated that the genes in question were closely related to several species of the Asteraceae family. Our data revealed that the G. bicolor PAL, CHS, DFR and ANS genes are closely related to the sequence of genes found in Chrysanthemum morifolium. Furthermore, the CHS, DFR and ANS of G. bicolor also tightly correlated with sequence from Callistephus chinensis. Further analysis also demonstrated that all conserved motifs contain active functional and catalytic domain supporting the flavonoid-related enzyme functions. All physicochemical parameters used in this study also meet the prediction criteria. The investigation of secondary structure prediction using two instruments (PSIPRED and SOPMA) revealed that all enzymes tested possess similar α-helix (H), β-sheet (E), and coil (C) composition. GbPAL showed a secondary structure dominated by helix (mainly alpha), GbCHS and GbDFR (Alpha-beta), and GbANS (mainly beta). Each enzyme was modeled, with RMSD values superimposed on the template: 0.125 Å (GbPAL), 0.061 Å (GbCHS), 0.065 Å (GbDFR), and 0.089 Å (GbANS). The 3D model evaluation was checked using four parameters, including PROCHECK, Verify3D, ERRAT, and QMEAN-Z score. All the parameters verified that the model showed a high quality (more than 90 % of the favored region), high ressemblence of the 3D and 1D amino acid sequence, the 3D modeles were acceptable since they possesed high resolution of the protein structure (2.5 – 3 Å), and the enzyme models have Z-Score values in the permissible range. The accuracy and reliability of the generated models met Ramachandran value in comparison to experimentally resolved protein templates. The molecular docking analysis showed that each substrate compound docked at the real binding site in every tested enzyme. Furthermore, they also showed similar results compared to other experimentally observed proteins. The overall findings provide an insight into the molecular characteristics of flavonoid-related genes, which might enrich our understanding for further genomic application of the Gynura bicolor.