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Experimental and in silico characterization of adenylosuccinate synthetase from patogen Helicobacter pylori

Helicobacter pylori is a Gram-negative microaerophilic bacterium that chronically colonizes the gastric epithelium. Presence of these bacteria in humans can lead to the development of several gastrointestinal diseases ; including non-symptomatic chronic gastritis, peptic ulcer disease, gastric mucosa- associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma. [1] Adenylosuccinate synthetase (ADSS) is one of the key enzymes in purine salvage pathway, which catalyses condensation reaction of IMP with L- aspartate (ASP) to form adenylosuccinate by GTP hydrolysis in the presence of Mg2+ ions. Bioinformatic studies showed that H. pylori lacks the genes for de novo synthesis of purines. Consequently, the viability of H. pylori relies on salvage pathway for purine synthesis. Thus, ADSS represents a potential drug target for H. pylori infection. [2-3] Using experimental methods, namely enzyme kinetics, we have successfully determined main kinetic parameters for all three substrates. Additionally, inhibition kinetics was performed with known inhibitor (hadacidin) and with reaction products adenylosuccinate and GDP. Preliminary binding experiments were conducted for all three substrates using microscale thermophoresis, in order to obtain binding parameters. Since attempts to solve 3D-structure of ADSS using crystallography were unsuccessful as yet, homology modelling was applied to obtain 3D-model. The model was constructed using multiple sequence alignment based on six closely related protein structures of ADSS. Several systems of ADSS enzyme alone, as well as in complex with different ligands, including substrates and inhibitors, were prepared for molecular dynamics (MD) simulations at the temperature of 310 K. Additionally, the in silico binding free energy experiments will be conducted for further characterization of modelled AdSS. The aim of this research is to decipher the mechanism of ADSS enzyme, with final goal of designing new inhibitors.

HTCC4 ; Protein ; Crystallography ; Biochemistry

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