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PDBsum entry 1oty

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protein ligands Protein-protein interface(s) links
Transferase PDB id
1oty
Jmol
Contents
Protein chains
237 a.a. *
Ligands
PO4 ×3
6MP ×3
Waters ×174
* Residue conservation analysis
PDB id:
1oty
Name: Transferase
Title: Native pnp +allo
Structure: Purine nucleoside phosphorylase. Chain: a, b, c. Synonym: inosine phosphorylase, pnp. Engineered: yes
Source: Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562
Biol. unit: Hexamer (from PDB file)
Resolution:
2.50Å     R-factor:   0.203     R-free:   0.236
Authors: S.E.Ealick,E.M.Bennett,R.Anand,J.A.Secrist,W.B.Parker,A.E.Ha P.W.Allan,D.T.Mcpherson,E.J.Sorscher
Key ref:
E.M.Bennett et al. (2003). Designer gene therapy using an Escherichia coli purine nucleoside phosphorylase/prodrug system. Chem Biol, 10, 1173-1181. PubMed id: 14700625 DOI: 10.1016/j.chembiol.2003.11.008
Date:
23-Mar-03     Release date:   17-Feb-04    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P0ABP8  (DEOD_ECOLI) -  Purine nucleoside phosphorylase DeoD-type
Seq:
Struc:
239 a.a.
237 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.2.4.2.1  - Purine-nucleoside phosphorylase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction:
1. Purine nucleoside + phosphate = purine + alpha-D-ribose 1-phosphate
2. Purine deoxynucleoside + phosphate = purine + 2'-deoxy-alpha-D-ribose 1-phosphate
Purine nucleoside
+
phosphate
Bound ligand (Het Group name = PO4)
corresponds exactly
=
purine
Bound ligand (Het Group name = 6MP)
matches with 90.00% similarity
+ alpha-D-ribose 1-phosphate
Purine deoxynucleoside
+ phosphate
= purine
+ 2'-deoxy-alpha-D-ribose 1-phosphate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     membrane   2 terms 
  Biological process     nucleobase-containing compound metabolic process   6 terms 
  Biochemical function     catalytic activity     6 terms  

 

 
    reference    
 
 
DOI no: 10.1016/j.chembiol.2003.11.008 Chem Biol 10:1173-1181 (2003)
PubMed id: 14700625  
 
 
Designer gene therapy using an Escherichia coli purine nucleoside phosphorylase/prodrug system.
E.M.Bennett, R.Anand, P.W.Allan, A.E.Hassan, J.S.Hong, D.N.Levasseur, D.T.McPherson, W.B.Parker, J.A.Secrist, E.J.Sorscher, T.M.Townes, W.R.Waud, S.E.Ealick.
 
  ABSTRACT  
 
Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.
 
  Selected figure(s)  
 
Figure 1.
Figure 1. Nucleosides and Their Interaction with the PNP Active Site(A) Active site structures for E. coli PNP and human PNP. Each active site is composed of eight segments. For each segment, the top line, in red, gives the human PNP residue numbers. The bottom line, in green, gives the E. coli PNP residue numbers. Segment eight in human PNP comes from an adjacent monomer, and segments two and eight in E. coli PNP come from an adjacent monomer (denoted by asterisks). The amino acid residues are aligned based on the structures of the two enzymes. The differences in active site residues result in different substrate specificity, even though the overall fold is the same.(B) Modified nucleosides used in this study. 1, 9-(2-deoxy-β-D-ribofuranosyl)-6-methylpurine (MeP-dR); 2, 9-(6-deoxy-α-L-talofuranosyl)-6-methylpurine [Me(talo)-MeP-R]; 3, 9-(6-deoxy-β-D-allofuranosyl)-6-methylpurine [Me(allo)-MeP-R]; 4, 9-α-L-lyxofuranosyl-adenine (lyxo-Ado); 5, 9-(5′-5′-di-C-methyl-β-D-ribofuranosyl)-6-methylpurine (5′,5′-dimethyl-MeP-R).
Figure 3.
Figure 3. Redesign of PNP to Accommodate a New Prodrug(A) Modeling studies predicted a steric clash (shown in green) between the 5′-methyl group of Me(talo)-MeP-R and the side chain of Met64.(B) Stereodiagram of the MeP-dR/wild-type PNP crystal structure (our unpublished data), with crystal structure carbon atoms shown in green. Modeling an additional carbon atom (shown in black) on C5′ results in unfavorable steric interactions (purple dotted lines) with the Met64 side chain.(C) The unfavorable interaction is eliminated in the M64V PNP.(D) Crystal structure of Me(talo)-MeP-R with M64V PNP, with the global minimum from computational docking overlayed in black wire frame representation. Panels (B) and (D) were created with Molscript [48] and Raster3D [49].
 
  The above figures are reprinted by permission from Cell Press: Chem Biol (2003, 10, 1173-1181) copyright 2003.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19343063 C.Hébrard, C.Dumontet, and L.P.Jordheim (2009).
Development of gene therapy in association with clinically used cytotoxic deoxynucleoside analogues.
  Cancer Gene Ther, 16, 541-550.  
17853921 C.S.Chen, Y.Jounaidi, T.Su, and D.J.Waxman (2007).
Enhancement of intratumoral cyclophosphamide pharmacokinetics and antitumor activity in a P450 2B11-based cancer gene therapy model.
  Cancer Gene Ther, 14, 935-944.  
17306358 D.Portsmouth, J.Hlavaty, and M.Renner (2007).
Suicide genes for cancer therapy.
  Mol Aspects Med, 28, 4.  
16307002 E.J.Sorscher, J.Harris, M.Alexander, A.Rottgers, K.Hardy, S.Ponnazhagan, J.F.Collawn, J.McClintock, C.D.Amsler, A.Webster, J.Maddry, B.J.Baker, and J.S.Hong (2006).
Activators of viral gene expression in polarized epithelial monolayers identified by rapid-throughput drug screening.
  Gene Ther, 13, 781-788.  
16859396 P.J.Russell, and A.Khatri (2006).
Novel gene-directed enzyme prodrug therapies against prostate cancer.
  Expert Opin Investig Drugs, 15, 947-961.  
15746571 G.U.Dachs, J.Tupper, and G.M.Tozer (2005).
From bench to bedside for gene-directed enzyme prodrug therapy of cancer.
  Anticancer Drugs, 16, 349-359.  
15983408 W.Bu, E.C.Settembre, M.H.el Kouni, and S.E.Ealick (2005).
Structural basis for inhibition of Escherichia coli uridine phosphorylase by 5-substituted acyclouridines.
  Acta Crystallogr D Biol Crystallogr, 61, 863-872.
PDB codes: 1u1c 1u1d 1u1e 1u1f 1u1g
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.