engleski

Biochemical characterization of His6-tagged adenylosuccinate synthetase from Helicobacter pylori: first step towards design of new inhibitors

Bacterium Helicobacter pylori is involved in development of several gastrointestinal diseases, including gastric cancer. Since H. pylori is estimated to infect ~50% of world’s population, and is increasingly evading currently used therapies, new ways of its eradication are urgently needed. Unlike most other bacteria, H. pylori does not have genes for enzymes involved in de novo purine synthesis. Adenylosuccinate synthetase (AdSS, EC 6.3.4.4) is one of the enzymes involved in purine recycling and is therefore critical for this bacterium’s survival. It catalyzes the synthesis of adenylosuccinate from IMP and aspartate, paired with hydrolysis of one phosphate group from GTP. Previously, we have purified and characterized native H. pylori AdSS (Bubić A et al. (2018) J Enz Inh Med Chem 33, 1405–1414), and here we present results obtained on His6-tagged variant of the enzyme, which is the first amongst bacterial AdSS enzymes. Nucleotide sequence coding for 6 His residues was inserted at the C-terminal end of the gene by inversed PCR. Enzyme was overexpressed in E. coli BL21-CodonPlus(DE3)-RIL cells, with IPTG induction. After two purification steps (affinity and size-exclusion chromatography), electrophoretically homogeneous AdSS-His6 was characterized by means of circular dichroism, enzyme kinetics measurement (regarding all three substrates) and stability (in respect to pH and temperature). Its properties were compared to those of native variant, and found to be very similar. For crystallization trials, AdSS-His6 was incubated with excess of IMP, GTP and hadacidin (inhibitor competitive towards aspartate). X-ray diffraction data from several crystals were collected at the synchrotron and 3D-structure solved by molecular replacement with Campylobacter jejuni AdSS (PDB kod 3R7T) as a model. Interactions in the active site were identified. Our findings represent first step towards design of new AdSS inhibitors, potential drugs for treatment of H. pylori infections.

Helicobacter pylori ; adenylosuccinate synthetase ; enzyme characterization ; protein 3D-structure ; active site interactions

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