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Bacterial purine nucleoside phosphorylase family of proteins

Purine nucleoside phosphorylases (PNPs) are the key enzymes of purine salvage. They catalyse the reversible phosphorolytic cleavage of the glycosidic bond of purine nucleosides and some analogues: beta-purine nucleoside + orthophosphate ↔ purine base + alpha-D-pentose-1-phosphate (1). Our scientific research is focused on studying complex catalytic mechanism of two PNPs from bacterial origin: Escherichia coli and very recently, from human pathogen Helicobacter pylori (HpPNP) whose purine methabolism is solely dependant on purine salvage (2). In contrast to human homologue, E. coli PNP has a broad substrate specificity which makes it a good candidate in gene therapy against solid tumours (3). Based on protein sequence analysis, HpPNP and E. coli PNP share 70 % similarity indicating their common catalytic mechanism. Hence, specific inhibitors of E. coli PNP may also inhibit HpPNP and in turn help in H. pylori eradication without affecting human host. Therefore, understanding the catalytic mechanism of these enzymes is of utmost importance. So far, we have conducted detailed kinetic studies and determined numerous 3D-structures of E. coli PNP binary and ternary complexes which enabled us to predict the main steps in its catalytic mechanism (4). Up to date we were able to grow crystals for X-ray studies only from conditions containing sulphate or phosphate ions. Since sulphate is an inhibitor and phosphate is a substrate of this enzyme, and, on the other hand, it is very difficult to distinguish these two ions in the electron density maps, new crystallisation conditions with no phosphate and/or sulphate are needed for further studies. For example, the crystals of ternary complex of E. coli PNP with phosphate and formycin A (structural analogue of adenosine) grown from highly concentrated ammonium phosphate (1.2 M) showed unusual phosphate binding mode (5) which could be an artefact and may not have biological relevance. For HpPNP only preliminary crystallisation experiments has been acomplished and its crystal structure is yet to be determined.

purine nucleoside phosphorylase; kinetics; 3D structures

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