Structural Basis for Substrate Specificity of Escherichia coli Purine Nucleoside Phosphorylase

Abstract

Purine nucleoside phosphorylase catalyzes reversible phosphorolysis of purine nucleosides and 2-deoxypurine nucleosides to the free base and ribose (or 2-deoxyribose) 1-phosphate. Whereas the human enzyme is specific for 6-oxopurine ribonucleosides, the Escherichia coli enzyme accepts additional substrates including 6-oxopurine ribonucleosides, 6-aminopurine ribonucleosides, and to a lesser extent purine arabinosides. These differences have been exploited in a potential suicide gene therapy treatment for solid tumors. In an effort to optimize this suicide gene therapy approach, we have determined the three-dimensional structure of the E. coli enzyme in complex with 10 nucleoside analogs and correlated the structures with kinetic measurements and computer modeling. These studies explain the preference of the enzyme for ribose sugars, show increased flexibility for active site residues Asp 204 and Arg 24 , and suggest that interactions involving the 1-and 6-positions of the purine and the 4-and 5-positions of the ribose provide the best opportunities to increase prodrug specificity and enzyme efficiency.

Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

“…The PfPNP sequence has greater similarity to E. coli than to human PNP (9), and its structure is hexameric as is the E. coli enzyme (7,20,21). PfPNP, however, has unique substrate specificity when compared with either E. coli or mammalian PNPs (9,21).…”

“…PfPNP, however, has unique substrate specificity when compared with either E. coli or mammalian PNPs (9,21). PfPNP is specific for 6-oxopurine rings in its substrates, nucleosides with adenine rings (including MTA) being neither substrates nor inhibitors for the enzyme.…”

“…We overexpressed and tested E. coli PNP, which has been extensively characterized structurally and enzymatically (20,21). E. coli PNP has activity against inosine and adenosine but has no detectable activity toward MTA or MTI.…”

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

“…The complex crystallized in a hexagonal space group P6 1 22 with only half of the hexamer in a asym- The active sites of monomers B and C ( Figure 6) contain two phosphate molecules each. Interestingly, none of these ions make previously observed hydrogen bonds 7,9,13,20,21 to the nucleoside and residues Gly20, Arg87, Ser90 and Arg43 from the neighbouring monomer. These two ions make hydrogen bonds to each other while the "standard" phosphate position is occupied by three water molecules.…”

TAT-mediated intracellular delivery of purine nucleoside phosphorylase corrects its deficiency in mice

“…Previously, E. coli PNP was shown to be active in mice (47). However, substrate specificities differ among PNPs of different species (48). Nevertheless, because of the greater than 85% amino acid homology between human and murine PNP, we anticipated that human PNP would also be active in mice.…”