PDBsum entry: 1oum

Transferase

PDB id: 1oum

Contents

Protein chains

237 a.a. *

Ligands

TAL ×3

PO4 ×2

Waters ×192

* Residue conservation analysis

PDB id:

1oum

Name:

Transferase

Title:

M64v pnp +talo

Structure:

Purine nucleoside phosphorylase. Chain: a, b, c. Synonym: inosine phosphorylase, pnp. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: deod or pup or b4384 or z5986 or ecs5343. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Hexamer (from PDB file)

Resolution:

2.40Å R-factor: 0.228 R-free: 0.260

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:

24-Mar-03 Release date: 17-Feb-04

Protein chains

P0ABP8  (DEOD_ECOLI) -  Purine nucleoside phosphorylase deoD-type

Seq: 239 a.a.

Struc: 237 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.4.2.1 - Purine-nucleoside phosphorylase.
• Reaction: Purine nucleoside + phosphate = purine + alpha-D-ribose 1-phosphate

Purine nucleoside (Bound ligand (Het Group name = TAL) matches with 90.00% similarity) + phosphate (Bound ligand (Het Group name = PO4) corresponds exactly) = purine + alpha-D-ribose 1-phosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Cellular component: membrane (2 terms)
• Biological process: nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (4 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.