Biocatalysts are intrinsically reactive and hence their operational stability is of vital significance for any bioprocess. The setback in biocatalyst stability has been tackled from diverse prospects. Inherently, stab...Biocatalysts are intrinsically reactive and hence their operational stability is of vital significance for any bioprocess. The setback in biocatalyst stability has been tackled from diverse prospects. Inherently, stable biocatalysts are markedly realized and a regular attempt is being made to seek out new organisms that harbor them. Here, we analyzed the industrial biocatalyst lipase A （Native） of Bacillus subtilis and its six thermostable mutants （2M, 3M, 4M, 6M, 9M and 12M） computationally using conformational sampling technique. Consequently, the various structural events deciphering thermostability like root mean square deviation, root mean square fluctuation, radius of gyration and polar surface area showed mutant 12M to be highly stable with statistical validation. Besides, static model analysis involving intra-molecular interactions, secondary structure, solvent accessibility, hydrogen bond pattern, simulated thermal denaturation and desolvation energy also supported 12M comparatively. Of note, the presence of high secondary structural rigidity and hydrogen bonds increased thermostability and functionality of 12M, thus selecting it as a best template for designing thermostable lipases in future. Also, this study has a significant implication toward a better understanding of conformational sampling in enzyme catalysis and enzyme engineering.展开更多
In this work, the most detrimental missense mutations of Madl protein that cause various types of cancer were identified computationally and the substrate binding efficiencies of those missense mutations were analyzed...In this work, the most detrimental missense mutations of Madl protein that cause various types of cancer were identified computationally and the substrate binding efficiencies of those missense mutations were analyzed. Out of 13 missense mutations, I Mutant 2.0, SIFT and PolyPhen programs identified 3 variants that were less stable, deleterious and damaging respectively. Subsequently, modeling of these 3 variants was performed to understand the change in their conformations with respect to the native Madl by computing their root mean squared deviation （RMSD）. Furthermore, the native protein and the 3 mutants were docked with the binding partner Mad2 to explain the substrate binding efficiencies of those detrimental missense mutations. The docking studies identified that all the 3 mutants caused lower binding affinity for Mad2 than the native protein. Finally, normal mode analysis determined that the loss of binding affinity of these 3 mutants was caused by altered flexibility in the amino acids that bind to Mad2 compared with the native protein. Thus, the present study showed that majority of the substrate binding amino acids in those 3 mutants displayed loss of flexibility, which could be the theoretical explanation of decreased binding affinity between the mutant Madl and Mad2.展开更多
文摘Biocatalysts are intrinsically reactive and hence their operational stability is of vital significance for any bioprocess. The setback in biocatalyst stability has been tackled from diverse prospects. Inherently, stable biocatalysts are markedly realized and a regular attempt is being made to seek out new organisms that harbor them. Here, we analyzed the industrial biocatalyst lipase A （Native） of Bacillus subtilis and its six thermostable mutants （2M, 3M, 4M, 6M, 9M and 12M） computationally using conformational sampling technique. Consequently, the various structural events deciphering thermostability like root mean square deviation, root mean square fluctuation, radius of gyration and polar surface area showed mutant 12M to be highly stable with statistical validation. Besides, static model analysis involving intra-molecular interactions, secondary structure, solvent accessibility, hydrogen bond pattern, simulated thermal denaturation and desolvation energy also supported 12M comparatively. Of note, the presence of high secondary structural rigidity and hydrogen bonds increased thermostability and functionality of 12M, thus selecting it as a best template for designing thermostable lipases in future. Also, this study has a significant implication toward a better understanding of conformational sampling in enzyme catalysis and enzyme engineering.
文摘In this work, the most detrimental missense mutations of Madl protein that cause various types of cancer were identified computationally and the substrate binding efficiencies of those missense mutations were analyzed. Out of 13 missense mutations, I Mutant 2.0, SIFT and PolyPhen programs identified 3 variants that were less stable, deleterious and damaging respectively. Subsequently, modeling of these 3 variants was performed to understand the change in their conformations with respect to the native Madl by computing their root mean squared deviation （RMSD）. Furthermore, the native protein and the 3 mutants were docked with the binding partner Mad2 to explain the substrate binding efficiencies of those detrimental missense mutations. The docking studies identified that all the 3 mutants caused lower binding affinity for Mad2 than the native protein. Finally, normal mode analysis determined that the loss of binding affinity of these 3 mutants was caused by altered flexibility in the amino acids that bind to Mad2 compared with the native protein. Thus, the present study showed that majority of the substrate binding amino acids in those 3 mutants displayed loss of flexibility, which could be the theoretical explanation of decreased binding affinity between the mutant Madl and Mad2.
