## 2 Results
### Docking Analysis  
To uncover possible functional differences between OsBADH2 and PaBADH2, protein docking analysis was carried out using BAD and GAB-ald as substrates (Figures 3 and 4). The three-dimensional structure of the PaBADH2 protein was developed and enzyme-substrate interaction was studied using protein docking where BAD was used as the substrate for BADH2. With GAB-ald, the interaction energy for a monomer of PaBADH2 and OsBADH2 was \(-\)3.3 kcal/mole and \(-\)3.2 kcal/mole, respectively, whereas, in the case of BAD, it was \(-\)5.1 kcal/mole and \(-\)5.2 kcal/mole, respectively. The conserved amino acids of the NAD+ catalytic domains are almost identical in both proteins. The active site of PaBADH2 for substrate binding comprised of, Ile158, Thr159, Pro160, Lys185, Ser187, Glu188, Phe236, Gly238, Ser239, and Thr242. Likewise, the active site of OsBADH2 consists of Glu297, Leu300, Phe301, Phe304, Trp305, Trp334, Ile338, and Arg349. From this analysis, interactions were found in the complexes between hydrogen bonds, ionic bonds, C-H bonds, and Van der Waals interactions. The BAD interacted with the active site of PaBADH2 via two hydrogen bonds (2.1 A\({}^{0}\)), two C-H bonds (3.49 A\({}^{0}\)), one ionic interaction (4.64 A\({}^{0}\)), and six Van der Waals interactions (3.16 A\({}^{0}\)). In this reaction, the hydroxyl group containing amino acids Ser239 and Thr242 from the active site of the enzyme were associated in hydrogen bond formation with a ligand molecule. Moreover, the nucleophilic nature of the tertiary amine of the substrate allows the formation of an ionic bond with Glu188 present in the active site of the enzyme. Thr159 and Thr237 were present close to the substrate forming a strong C-H bond. Other than these, Phe236, Gly238, Lys185, Ser187, and Ile158 were involved in the Van der Waals interaction. In the same way, OsBADH2 also contained Glu297 that formed ionic interaction with the ligand, whereas other amino acids such as Leu300, Phe301, Phe304, Trp305, Trp334, Ile338, Arg349 were engaged in Van der Waals bond formation with the substrate. Overall, the docking analysis of both PaBADH2 and OsBADH2 suggested that there was not much difference in the interaction energies and the bond formation in the active site of both the enzymes; hence, it can be said that the PaBADH2 enzyme is likely to have similar activities.  
Figure 4: Docking analysis of PaBADH2 with 4-amino butaraldehyde (GAB-ald): (**a**) docked 3D structure of PaBADH2-GAB-ald complex; (**b**) showing H-bond donor (pink) and acceptor (green) surfaces in PaBADH2-GAB-ald complex; (**c**) the 2D view of interacting residues of PaBADH2- GAB-ald complex at ligand binding site that involves H-bond (green circle) and Van der Waals bond (light green circle); (**d**) docked 3D structure of OsBADH2 GAB-ald complex; (**e**) showing H-bond donor (pink) and acceptor (green) surfaces in OsBADH2 GAB-ald complex; (**f**) the 2D view of interacting residues of OsBADH2-GAB-ald complex at ligand binding site that involves H-bonds (green circle) and Van der Waals bonds (light green circle).  
Figure 3: Docking analysis of PaBADH2 with betaine aldehyde (BAD): (**a**) docked 3D structure of PaBADH2-BAD complex; (**b**) showing H-bond donor (pink) and acceptor (green) surfaces in PaBADH2-BAD complex; (**c**) the 2D view of interacting residues of PaBADH2-BAD complex at ligand binding site that involves H-bond (green circle), Van der Waals bonds (light green circle), and electrostatic interaction (brown circle).