Steve Ealick's Research Group
Escherichia coli Purine Nucleoside Phosphorylase
PDB file: 1ECP1 (original paper)
1PK7# (native complexed with adenosine and sulfate/phosphate)
1PK9# (native complexed with 2-fluoroadenosine and sulfate/phosphate)
1PKE# (native complexed with 2-fluoro-2′-deoxyadenosine and sulfate/phosphate)
1PR0# (native complexed with inosine and phosphate/sulfate)
1PR1# (native complexed with formycin B and phosphate/sulfate)
1PR2# (native complexed with 9-β-D-[2-deoxyribofuranosyl]-6-methylpurine and phosphate/sulfate)
1PR4# (native complexed with 9-β-D-ribofuranosyl-6-methylthiopurine and phosphate/sulfate)
1PR5# (native complexed with 7-deazaadenosine and phosphate/sulfate)
1PR6# (native complexed with 9-β-D-xylofuranosyladenine and phosphate/sulfate)
1PW7# (native complexed with 9-β-D-arabinofuranosyladenine and sulfate/phosphate)
1OTY* (native PNP complexed with 9-(6-deoxy-b-D-allofuranosyl)-6-methylpurine (Me(allo)-MeP-R))
1OTX* (M64V PNP)
1OU4* (native PNP complexed with 9-(6-deoxy-α-L-talofuranosyl)-6-methylpurine (Me(talo)-MeP-R))
1OUM* (M64V PNP complexed with 9-(6-deoxy-α-L-talofuranosyl)-6-methylpurine (Me(talo)-MeP-R))
1OV6* (M64V PNP complexed with 9-(6-deoxy-β-D-allofuranosyl)-6-methylpurine (Me(allo)-MeP-R))
1OVG* (M64V PNP complexed with9-(2-deoxy-β-D-ribofuranosyl)-6-methylpurine (MeP-dR))
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of (2′-deoxy)purine ribonucleosides to free base and (2′-deoxy)ribose-1-phosphate. The monomer unit in mammals has a molecular weight of about 31 kDa, while the monomer in prokaryotes is about 26 kDa. The active sites of E. coli and human PNP have different amino acids, giving them different specificity and different inhibition profiles. E. coli PNP will accept 6-amino and 6-oxopurine (2′-deoxy)purine ribonucleosides to free base, while mammlian PNP only accepts the latter as a substrate. The differences in enzyme specificity offer possibilities for gene therapy for treatment of tumors.
The active enzyme in E.coli is hexameric, while in mammals it is trimeric. The E. coli PNP hexamer can be considered to be a "trimer of dimers", with contacts between subunits within dimers that are more extensive than those between the three dimers. Two adjacent subunits are involved in the formation of each active site. The active site in hexameric PNP is more accessible than that in trimeric PNP. Compare this structure to the monomers of human PNP and bovine PNP.
Click the image to enlarge.
This schematic diagram shows the phosphate binding site, while this one shows residues at the purine binding site.
1Mao C, Cook WJ, Zhou M, Koszalka GW, Krenitsky TA, and Ealick SE. The Crystal Structure of Escherichia coli Purine Nucleoside Phosphorylase: A Comparison with the Human Enzyme Reveals a Conserved Topology. Structure 5:1373-1383 (1997).
Cook WJ, Ealick SE, Krenitsky TA, Stoeckler JD, Helliwell JR, and Bugg CE. Crystallization and Preliminary X-ray Investigation of Purine Nucleoside Phosphorylase from Escherichia coli. J. Biol. Chem. 260(24):12968-12969 (1985).
#Bennett EM, Li C, Allan PW, Parker WB and Ealick SE. Structural Basis for Substrate Specificity of Escherichia coli Purine Nucleoside Phosphorylase. J. Biol. Chem. 278:47110-47118 (2003).
*Bennett EM, Anand R, Allan PW, Hassan AEA, Hong JS, Levasseur DN, McPherson DT, Parker WB, Secrist JA III, Sorscher EJ, Townes TM, Waud WR and Ealick SE. Designer Gene Therapy Using an Escherichia coli Purine Nucleoside Phosphorylase/Prodrug System. Chem. Biol. 10:1173-1181 (2003).