## 3 Results and Discussion
### Molecular Dynamics Simulation Analysis
As can be seen, the hydrogen bonds to Asp203 and His600 (see Figure 4A) were broken, while the hydrogen bond with Asp327 was formed again after a momentary break, and remained stable during the whole simulation. This rearrangement could explain the shift in the RMSD of ACA (black curve, Figure 6B). In the case of LUT, the highest occupancy with Asp443 (99.24%) showed that it stayed bonded to this residue during the whole MD simulation, and the occupancies corresponding to residues Asp542 and Glu300 (21.12% and 11.80%, respectively) explain why the RMSD of LUT was shifting between two different values (blue curve, Figure 6B). In particular, LUT was forming and breaking hydrogen bonds with these residues intermittently. MAY ligand showed a more varied behavior. The hydrogen bond with Thr205 predicted by molecular docking disappeared during the MD simulation. Moreover, the one with Asp327 (89.11%) remained during almost the whole simulation. Furthermore, MAY seems to intermittently form and break hydrogen bonds with the rest of the Asp residues present in the active site. The occupancies are 60.08%, 17.48%, and 4.07% with Asp203, Asp542, and Asp443, respectively. Additionally, MAY forms a hydrogen bond with Glu300 (24.59%), similar to that observed for LUT with 11.80%. Figure 6: RMSD values of MD simulations (**A**) for the atoms of the complexes of acarbose (ACA), maysin (MAY), and luteolin (LUT) with maltase-glucoamylase (NtMGAM) (**B**) RMSD variations after 125 ns calculated over the atoms of the ligands. The distance distributions and the distance versus time (from residue to ligand) are plotted in Figure 7, in order to explain the MAY occupancies, which present more variations compared to ACA occupancies. Distance distributions (Figure 7A,C) for MAY showed more peaks, which means that MAY was oscillating between these residues (forming and breaking its hydrogen bonds). However, when the distance versus time is analyzed (Figure 7B,D), a better understanding of the involved dynamics is observed, specifically, if the ligands remained in the same place inside the active site or if they were moving away. In the case of MAY, it is clear that MAY was moving away from residues Asp443, Asp203, and Asp542, which are the Asp residues more internal in the active site. In addition, it was getting closer to Asp327 and Glu300, which are the residues located at the outermost part of the active site, contrary to ACA whose distances to the residues remained very stable. The lower stability of the NtMGAM-MAY complex can be understood with an analysis of the water behavior inside the active site pocket. Figure 8 shows the number of water molecules within a distance of 6 A to the residue Asp443, which is located at the bottom of the active site pocket. For the NtMGAM-LUT complex, the amount of water inside the pocket was very close to that of the protein alone (without a ligand). In the case of MAY, the amount of water increased considerably around 50 ns of the simulation. The inclusion of a larger amount of water in the active site decreased the hydrophobicity, affecting the protein-ligand complex stability. Thus, the variations of the RMSD around 50 ns of the \begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{3}{c}{**Occupancy (\%)**} \\ \hline Amino acids & ACA & LUT & MAY \\ Asp443 & 98.30 & 99.24 & 4.07 \\ Asp327 & 81.09 & 0.00 & 89.11 \\ Asp203 & 0.00 & 0.18 & 60.08 \\ Asp542 & 3.56 & 21.12 & 17.48 \\ Glu300 & 0.00 & 11.80 & 24.59 \\ His600 & 0.00 & 0.00 & 0.00 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Hydrogen bond occupancies of the MD trajectories in ACA, MAY, and LUT with amino acids of the active site. Figure 7: Distribution of residues-ligand distances in (**A**) MAY and (**C**) ACA and distance versus simulation time of (**B**) MAY and (**D**) ACA. NtMGAM-MAY complex (red curve, Figure 6A) could be explained for the hydrophilic character of MAY. For ACA, the amount of water in the complex compared to that in the protein alone decreased considerably along the simulated time.