Does molecular docking reveal alternative chemopreventive mechanism of activation of oxidoreductase by sulforaphane isothiocyanates?

Abstract

Isothiocyanates (ITC) are well-known chemopreventive agents extracted from vegetables. This activity results from the activation of human oxidoreductase. In this letter, the uncompetitive activatory mechanism of ITC was investigated using docking and molecular dynamics simulations. This indicates that NAD(P)H:quinone oxidoreductase can efficiently improve enzyme-substrate recognition within the catalytic site if the ITC activator supports the interaction in the uncompetitive binding site.Response to Reviewers: Answers for reviews:Reviewer #2: The authors have explored uncompetitive activatory mechanism of isothiocyanates using docking and molecular dynamics simulations. Though the methodology adopted appears to be sound and discussion is convincing, how can they validate their hypothesis (either theoretical or experimental)? Considering the suggestions we performed additional analyses to validate the hypothesis. It seems that the movements of several amino acids in the USB are constrained by the presence of the activator resulting in some changes of the CBS. Further testing of this hypothesis has been performed using the reduced dynamic approach (RedMD), which is based on the coarse-grained model of the protein. Thus, two representations of NAD(P)H:quinine oxidoreductase have been prepared by changing each amino acid with artificial alpha carbon atoms having appropriate properties to fake replaced amino acids. The first representation mimics the original 1D4A crystal structure, whereas the second one modifies the properties of ASN45÷47 of chain B and D, respectively. Generally, it reflects the constrains caused by the presence of activators in the UBS. Moreover, the applied modifications resulted in the increase of masses and harmonic constant. Afterward, both representations have been used in Langevin's dynamics simulations repeated 10 times with different random seeds. The whole trajectory time was 20 ns with temperature of 300K. The atomic coordinates of the system were sampled every 20fs. Then, the entire set of the obtained frames has been superimposed by ASN45÷47 of chain B and D, respectively.Fig. 5 in the revised version represents the resulting histograms of RMSD of alpha carbon (CA) of TYR128 calculated against its reference position taken from the original 1D4A file for the whole trajectory. The red color indicates TYR128 CA of the 1D4A reference structure, while the blue one shows TYR128 CA of the modified representations. Taking into account the imposed constraints on residues 45÷47 minor but noticeable change of TYR128 CA behavior during the simulations might be noticed. The detailed analysis of the histogram revealed that TYR128 CA occupied approximately 5% more time in the mean aberration. Additionally, it seems that TYR128 is less prone to the high deviation as it is shown in the subplot with logarithmic scale. The above conclusion might prove our previous observations. We observed that the constraints of 45÷47 residues either by the presence of the activators or by manual modification...

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