Homology Modeling Of β-Glucuronidases From E. Coli and T. Maritima

The enzyme β-Glucuronidases (GUS) which belongs to the glycoside hydrolase family of enzymes, can hydrolyze any aglycne conjugated to D-glucuronic acid through a β-O~glycosidic linkage. It is present in almost all tissues of vertebrates and their residentinal flora, including E. coli. However, GUS enzymes obtained from different sources have different stability towards heat, resistance to detergents and varying catalytic activities. A good understanding or the reasons for this variation can lead to designing new enzymes with desired level of property. having great prospect in the industry. For this purpose, studies on the three-dimensional structure of GUS enzyme can offer insights on the structure-function correlations, and provide information on the distribution or certain residues both in E. coli and T. maritima enzymes. The structures of GUS enzymes from E. coli and T. maritima are not known experimentally. As such in the current work, homology modeling or the three-dimensional structure of both variants of the GUS enzyme was carried out based on the solved crystal structure of Human GUS enzyme. Multiple sequence alignment for both enzyme sequences was carried out in order to locate the most suitable template for homology modeling and the models thus prepared were found to cotain 32-43% sequence identity with the template. Superposition of the model obtained with the template as well as structural alignment were carried out to classify the structural differences. This paper will also present an analysis and verification studies of the model based on various criteria. The current work offers a better understanding of the structural differences between GUS enzymes from different sources, as well as suggests regions for further modification using experimental and computational methods

Molecular dynamics studies of human β-Glucuronidase

Problem statement: The enzyme β-glucuronidase is being used as a reporter molecule in the area of genetic engineering, as a component of prodrug therapy in cancer treatment and in the scouring process of cotton fabrics. However, a detailed understanding of the factors responsible for the stability and the activity of this enzyme is still not available. Molecular Dynamics (MD) simulations provide an estimate of equilibrium and dynamic properties of enzyme systems that cannot be calculated analytically. With this perspective, molecular dynamics simulations of human β- glucuronidase (GUS) have been carried out to determine the behavior of this enzyme in vacuum and solvent environments at a defined temperature. Approach: CHARMM force field along with distance dependent dielectric model was used to represent the solvent environment in the MD simulations. The parameters employed in various stages of MD simulations had been selected based on repeated trials under various conditions as a method of choosing the optimum parameters for each stage. Results: It was found that simulations in vacuum caused the backbone of GUS to have smaller fluctuations from their mean values compared with the fluctuation in implicit solvent simulations, due to the fact that vacuum environment did not provide for the electrostatic interactions affecting the backbone of GUS that may otherwise exist in a solvent environment. Conclusion: Inclusion of solvent effects in MD simulations is crucial in understanding structural flexibility and stability of β-glucuronidase. Implicit solvent method can provide a realistic inclusion of backbone flexibility and structural compactness of GUS, which will have profound influence on the stability and activity of the enzymes, with a marginal increase in computational time

In silico designing of thermostabe β-Glucuronidase (GUS)

This research has used Molecular Dynamics (MD) techniquess as an in silico method of correlating the experimental studies done on GUS enzyme with computational study and has analyzed and identified the structural factors responsible for thermostability of this enzyme. GUS from E. coli is heat labile and inhibited by detergents and products, which hinder its usefulness as a reporter molecules in genetic engineering. Therefore a more thermostable GUS enzyme needs to be designed for industrial applications. Using homology modeling, structures of mesophilic and thermophilic GUS enzymes from E. coli and T. maritima have been constructed based on the crystal structure of human GUS enzyme. MD simulations of these mesophilic and thermophilic GUS enzymes at temperatures of 300 K and 353 K in vacuum and implicit solvent have provided information on thermolabile regions in the enzymatic structure to be targeted for thermal stability. The RMS deviation of backbone atoms and helical residues from their initial coordinates was analysed for the resulting simulation trajectories. A higher number of charged residues found in the thermostable GUS were found to be responsible for stability of the helices compared to mesophilic GUS. From analysis of salt bridges, the presence of higher number of Glu-Arg and Glu-Lys salt bridge pairs were found to be responsible for be responsible for thermostability of T.maritima GUS The thermolabile residues 150-155 in wild type E. coli GUS structures were identified, and have been suggested as mutation points for experimental studies to improve thermostability. These residues have not been identified before, and are suggested to be replaced with Ala, Arg, Glu, and Lys. The choice of Ala and Arg are supported by previous experimental mutations in other regions of GUS and have resulted in thermostable GUS enzymes