Structural and biophysical characterization of cassava linamarase and the role of solvents on linamarin’s properties: a computational study

Abstract

Linamarase and linamarin mainly from cassava have many applications ranging from food, environmental to the medical industry. To better explore the potential of this enzyme and its substrate, one needs to understand its interaction mechanism at the molecular and atomistic level. In this thesis, the three‒dimensional (3D) structure of linamarase was built via homology modeling. The developed model was used to determine the binding orientation and mechanism of linamarin to the enzyme using molecular docking. Molecular dynamics simulation was used to determine the stability of the built model and when complexed with the ligand. It was interesting to note that complex 1 with the low binding‒free energy of ‒6.9 kcal/mol showed a larger Root Mean Square Deviation (RMSD) value with two maxima at 0.255 and 0.310 nm compared to complex 2 with the best binding‒free energy of ‒7.2 kcal/mol, whose RMSD value shows the maxima at 0.19 nm. The end‒point free energy method based on Molecular Mechanics Poisson Boltzmann Surface Area (MM/PBSA) was used to rescored binding free energy obtained from docking calculations. The ensemble structure was observed to be relatively stable compared to the modelled structure. Furthermore, the stability and conformational orientation preferences of linamarin in different solvents was established using classical molecular dynamics, and found to be solvent dependent. The effects of solvents on the stability and conformational preference is pronounced by different probability density maxima of the measured reaction coordinate/properties. Linamarin is observed to be stable in methanol followed by dimethyl sulfoxide (DMSO) and least stable in water. Solvent polarity was observed to influence the stability and conformation preference of the title compound. Linamarin exists in trans and gauche conformations, the former was observed to be more stable in water than other solvents and the latter in DMSO. The measured reaction coordinates, distance and dihedral angles ascertained that the conformational preference is due to rotation at 𝜙 = ± 180o and 𝜙 = ± 50o . Finally, the stability of linamarin was also attributed to different numbers of inter and intra hydrogen bonds formed in different solvents. Results presented in this thesis provides atomistic insights on the role of solvents polarity on linamarase‒linamarin complex interaction and stability. The findings provide important information on the application of linamarase and linamarin in different fields including food processing and in drugs.