## 3 Discussion
### Docking Analysis  
The variations in amino acid sequence were further studied by structural characterization of PaBADH2 protein through docking analysis. A comparative PaBADH2 and OsBADH2 homology modeling showed that the alignment of both models resulted in a 0.115 RMSD value. That claims that the 3D structures of both the proteins are closely similar without any major differences. That shows changes in amino acid sequence do not alter the 3D structure of the protein. To verify that it does not hamper the interaction between protein and substrate, docking analyses were carried out. In BADH2 enzymes, the NAD+ binding domain is mainly responsible for the conversion of intermediate aldehydes to their respective carboxylic acids. The crystal structure of AMADH from _Spinacia olerca_, _Pisum sativum_[26], and _Oryza sativa_[27] have been studied. The 3D structure and simulation studies of OsBADH2 enzyme carried out by [PERSON], [PERSON], and [PERSON] [27] inferred that Asn162, Glu260, and Cys294 are prominent amino acids of the catalytic domains. The previous study in rice suggested this catalytic triad as crucial for the catalytic activity of ALDH. These three amino acids are conserved in many plant species. In the hemithioacetal enzyme formation, Cys294 and Glu260 are involved and Asn162 helps in the stabilization of an intermediate. In the protein-ligand interaction, the oligomerization domain shows little mobility, while during the interaction, the NAD+ domain shows maximum mobility. Asn162 and Glu260 form strong hydrogen bonds with nearby residues, resulting in a rigid conformation. The strong interaction network may help to shape a suitable cavity size and dimension that fits both substrate and cofactor. In contrast, Cys294 remained mobile inside the pocket. Numerous reports are available that show that mutation in any of the amino acids from this catalytic domain results in reduced enzyme-substrate affinity. The conversion of Asn162 to alanine resulted in a momentous reduction in enzyme affinity with the substrate, while a change of Glu260 to alanine led to a complete loss of enzyme activity [15; 28; 29]. Mutation in Cys294 to alanine was reported to entirely abolish the activity of the enzyme [13; 14]. [PERSON] and [PERSON] [30] executed a comparative rice BADH2 enzyme interaction study using rice mutant lines BADH2N162A, BADH2E2E60A, and BADH2C294A. The docking analysis revealed that the protein structure developed by mutant lines showed a narrower binding pocket than the wild-type BADH2 protein, and it also exhibited lower interaction energy when docked with GAB-ald. The minimum enzyme interaction was found in BADH2N162A, followed by BADH2E260A and BADH2C294A. Therefore, it was concluded that these conserved amino acids play a major role in the establishment of appropriate binding cavity for better accommodation of the substrate. Any further changes in these conserved amino acids lead to change in the dynamics of the binding cavity, which, in turn, affects the enzyme-substrate interaction. [PERSON], [PERSON], [PERSON] and [PERSON] [20] reported docking analysis in which they showed E260 as a conserved amino acid of substrate binding pocket; however, it is missing in their PND BADH2 sequence. The same amino acid is present in OsBADH2 and PaBADH2 sequences. Further, in the sequence, they reported glycine in place of glutamate (E260). This questions the authenticity of their sequence. In contrast, the docking analysis of our sequence confirms the functionality of PaBADH2. The high PaBADH2 enzyme activity in vivo (46 nM/min/g) confirmed these findings. [PERSON] et al. [31] reported BADH2 enzyme activity in aromatic (Khao Dawk Mali 105) and nonaromatic rice (Sew-Mae- Pah-Tawng) at 26 nmol/min and 37 nmol/min, respectively. These results revealed higher BADH2 activity in P. amaryllifolius than in rice. GAB-ald and BAD are specific substrates of BADH2. [PERSON] et al. [32] have also reported BADH2 activity in barley, wheat, and spinach as 0.17, 0.22, and 0.82 nM/min/mg of total protein, respectively.