Abstract
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.
Highlights
Human β-glucuronidase belongs to family 2 glycosyl hydrolases that cleave β-D-glucuronic acid residues from the non reducing termini of glycosaminoglycans
System Preparation: In order to prepare a sample for Molecular Dynamics (MD) simulation, a model system consisting of N particles is selected and Newton's equations of motion are solved for this system until the properties of the system reach a stable value, or more appropriately reach equilibration
The Chemistry at HARvard Macromolecular Mechanics (CHARMM) program was used for performing all the molecular dynamics simulations
Summary
Human β-glucuronidase belongs to family 2 glycosyl hydrolases that cleave β-D-glucuronic acid residues from the non reducing termini of glycosaminoglycans. The X-ray structure of a human β-glucuronidase has been determined, which shows it to be a tetramer (Jain et al, 1996), with each monomer 653 amino acid residues long and a size of 80 kDa. It was revealed that each monomer of the human GUS structure has three structural domains, namely the jelly roll barrel, immunoglobulin constant domain and a Triosephosphateisomerase (TIM) barrel, in addition to it having an overall fold similarity to the N-terminal region of E. coli β-galactosidase. The active site of human β-glucuronidase has been based on the experimentally determined active site in E. coli βgalactosidase and corresponds to the amino acid residues Glu451, Glu540 and Tyr504. The nature of the active site residues have been discussed previously (Islam et al, 1999)
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