Purine salvage pathway enzymes – structure, inhibitors, mechanism and protein-protein interactions

Keywords

purine nucleoside phosphorylase
adenylosuccinate synthetase
mechanism of catalysis
three dimensional structure inhibitors
protein-protein interactions
X-ray crystallography
Analytical ultracentrifugation
Spectroscopy
New therapies
Purine salvage pathway

Research description

A. Molecular mechanism of cooperation of subunits in hexameric purine nucleoside phosphorylase (PNP)

Gradual loss of a catalytic activity towards natural substrates of hexameric PNP from E. coli does not correlate with the loss of the ability to bind ligands, substrates, and inhibitors. The values of dissociation constants characterizing ligand binding depend on the specific activity of the enzyme used in the experiment. Furthermore, there is a stable state of the enzyme, in which it is no longer able to catalyse reaction with natural substrates, but can still catalyse the same reaction if a substrate resembling the transition state is used. The active site conformations of individual subunits in the X-ray structure of this hexameric molecule reflect the presence of intermediate states observed in the enzyme activity decline profiles. Our goal is to elucidate the molecular mechanism of enzyme subunits cooperation, its role in catalysis, and characterize the intermediate states.

B. Adenylosuccinate synthetase (AdSS) and purine nucleoside phosphorylase (PNP) as new molecular targets in eradication of H. pylori

The aim of the current project is to search for potent selective inhibitors of purine nucleoside phosphorylase and adenylosuccinate synthetase from H. pylori, because in our previous studies we showed that inhibitors of these two enzymes, those that effectively penetrate the interior of the cell of this bacterium, show bacteriostatic, and some also  actericidal properties against reference strains, and strains resistant to clarithromycin, metronidazole and both of these antibiotics.

C. Protein-protein interactions of purine salvage pathway in H. pylori

We are just starting a project, in collaboration with Dr. Ivana Leščić Ašler z Ruđer Bošković Institute, to characterize protein-protein interactions (PPIs) in the purine salvage pathway in H. pylori. It is estimated that about 80% of proteins function in a stable or transient complexes, hence investigating PPIs is essential for understanding physiology of this pathogen, while PPIs of purine salvage pathway enzymes in H. pylori are poorly investigated so far.