Still a Long Way to Fully Understanding the Molecular Mechanism of Escherichia coli Purine Nucleoside Phosphorylase

Abstract

Abstract. The results of several decades of studying the catalytic mechanism of Escherichia coli purine nucleoside phosphorylases (PNP) by solution studies and crystal structure determinations are presented. Potentially PNPs can be used for enzyme-activating prodrug gene therapy against solid tumours because of the differences in specificity between human and E. coli PNPs. Biologically active form of PNP from E. coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. Two conformations of the active site are possible after substrate(s) binding: open and closed. A series of determined 3D-structures of PNP binary and ternary complexes facilitated the prediction of the main steps in the catalytic mechanism. For their validation the active site mutants: Arg24Ala, Asp204Ala, Arg217Ala, Asp204Asn and double mutant Asp204Ala/Arg217Ala were prepared. The activity tests confirm that catalysis involves protonation of the purine base at position N7 and give better insight into the cooperativity between subunits in this oligomeric enzyme. (doi: 10.5562/cca2116)

New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined H/D Exchange Approach and Molecular Dynamics Simulations

“…Negative, doubly charged phosphate anion rearranges, upon binding, Hbonds, and attracts positively charged residues affecting dynamics of protein backbone. In this context, the use of Arg24Ala mutant in this work confirmed previous knowledge [19][20][21][22][23][24] on the key role of Arg24 in the PNP catalysis.…”

“…This remarkable enzyme has been the subject of our scientific interest for many years [19][20][21][22][23][24]. PNPs are versatile catalysts of the reversible phosphorolysis of purine (2′-deoxy)-ribonucleosides [25,26]: purine (2′-deoxy)-ribonucleoside + orthophosphate ↔ purine base + (2′-deoxy)-ribose 1-phosphate Due to their catalytic function and much broader specificity compared with their trimeric human counterpart [26], hexameric PNPs (e.g., from E. coli) have been investigated for the efficient synthesis of nucleoside analogues [27] as well as for the activation of pro-drugs in anti-cancer gene therapies [28].…”

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features

“…For many years we have been studying a homohexameric enzyme purine nucleoside phosphorylase (PNP, purine nucleoside orthophosphate ribosyl transferase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2.4.2.1.html) and the conformational changes of its six active sites upon ligand binding, with the final goal of understanding the highly complex mechanism of this enzyme action . PNP catalyses the reversible phosphorolytic cleavage of the glycosidic bond of purine ribo‐ and deoxyribonucleosides:…”

Can Crystal Symmetry and Packing Influence the Active Site Conformation of Homohexameric Purine Nucleoside Phosphorylases?

“…[6] It is interesting to stress out, that in the case with 3 open + 3 closed active site conformations they alternate regularly, while in the case of 4 open and 2 closed active sites, the closed sites are always next to each other and belong to two different dimers of one homohexamer.…”