Purine-nucleoside phosphorylase

 

Purine metabolism in the parasite Plasmodium has been identified as a promising target for antimalarial therapies due to slight alterations around the active site when compared to mammalian purine nucleoside phosphorylase (PNP). PNP is part of a salvage pathway for the biosynthesis of purines, which are essential to parasite survival due to location of the organism, erthrocytes and Plasmodium itself not being able to produce purines de novo .

 

Reference Protein and Structure

Sequence
Q8I3X4 UniProt (2.4.2.1) IPR000845 (Sequence Homologues) (PDB Homologues)
Biological species
Plasmodium falciparum 3D7 (malaria parasite P. falciparum) Uniprot
PDB
2bsx - Crystal structure of the Plasmodium falciparum purine nucleoside phosphorylase complexed with inosine (2.0 Å) PDBe PDBsum 2bsx
Catalytic CATH Domains
3.40.50.1580 CATHdb (see all for 2bsx)
Click To Show Structure

Enzyme Reaction (EC:2.4.2.1)

inosine
CHEBI:17596ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
→
alpha-D-ribose 1-phosphate(2-)
CHEBI:57720ChEBI
+
hypoxanthine
CHEBI:17368ChEBI
Alternative enzyme names: PNPase, PUNPI, PUNPII, Inosine phosphorylase, Inosine-guanosine phosphorylase, Nucleotide phosphatase, Purine deoxynucleoside phosphorylase, Purine deoxyribonucleoside phosphorylase, Purine nucleoside phosphorylase, Purine ribonucleoside phosphorylase,

Enzyme Mechanism

Introduction

PNP is proposed to partake in a Sn1 nucleophilic substitution upon activation from conformational changes in the active site loop. It's substrate is inosine that has formed from prior deamination of adenosine by adenosine deaminase. Asp206 protonates N7 on the inosine group which facilitates glycosidic bond cleavage while Arg27 (alongside Arg45 and Arg88) stabilise the incoming oxygen nucleophile on a phosphate group. The reaction sees the formation of an oxocarbenium ion transition state which collapses to form ribose-phosphate and hypoxanthine which is a precursor to purine biosynthesis required by the parasite.

Catalytic Residues Roles

UniProt PDB* (2bsx)
Arg27 Arg27A Stabilises thus activates the phosphate anion for nucleophilic attack. electrostatic stabiliser
Asp206 Asp206A Provides a proton to N7 of the purine as the glycosidic bond cleaves. proton acceptor, proton donor
Arg88, Arg45 Arg88A, Arg45A(AD) Bind to and stabilise the phosphate ion via hydrogen bonds. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

heterolysis, charge delocalisation, proton transfer, elimination (not covered by the Ingold mechanisms), intermediate formation, overall reactant used, bimolecular nucleophilic addition, intermediate terminated, inferred reaction step, native state of enzyme regenerated, overall product formed

References

  1. Chaikuad A et al. (2009), BMC Struct Biol, 9, 42-. Conservation of structure and activity in Plasmodium purine nucleoside phosphorylases. DOI:10.1186/1472-6807-9-42. PMID:19575810.
  2. Madrid DC et al. (2008), J Biol Chem, 283, 35899-35907. Plasmodium falciparum purine nucleoside phosphorylase is critical for viability of malaria parasites. DOI:10.1074/jbc.M807218200. PMID:18957439.
  3. Schnick C et al. (2005), Acta Crystallogr D Biol Crystallogr, 61, 1245-1254. Structures ofPlasmodium falciparumpurine nucleoside phosphorylase complexed with sulfate and its natural substrate inosine. DOI:10.1107/s0907444905020251. PMID:16131758.
  4. Tahirov TH et al. (2004), J Mol Biol, 337, 1149-1160. Crystal Structure of Purine Nucleoside Phosphorylase from Thermus thermophilus. DOI:10.1016/j.jmb.2004.02.016. PMID:15046984.
  5. Bennett EM et al. (2003), J Biol Chem, 278, 47110-47118. Structural Basis for Substrate Specificity of Escherichia coli Purine Nucleoside Phosphorylase. DOI:10.1074/jbc.m304622200. PMID:12937174.

Step 1. Asp206 protonates the inosine base, promoting electron withdrawal to cleave the glycosidic bond.

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Catalytic Residues Roles

Residue Roles
Arg27A electrostatic stabiliser
Arg45A(AD) electrostatic stabiliser
Arg88A electrostatic stabiliser
Asp206A proton donor

Chemical Components

heterolysis, charge delocalisation, proton transfer, elimination (not covered by the Ingold mechanisms), intermediate formation, overall reactant used

Step 2. Arg27 helps activate the phosphate group for nucleophilic attack on a carbon on the ribose ring by stabilising the negatively charged group.

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Catalytic Residues Roles

Residue Roles
Arg27A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, intermediate terminated

Step 3. The ribose-phosphate is deprotonated by Asp206.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp206A proton acceptor

Chemical Components

inferred reaction step, proton transfer, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. Asp206 protonates the inosine base, promoting electron withdrawal to cleave the glycosidic bond.

Step 2. Arg27 helps activate the phosphate group for nucleophilic attack on a carbon on the ribose ring by stabilising the negatively charged group.

Step 3. The ribose-phosphate is deprotonated by Asp206.

Products.

Contributors

Gemma L. Holliday, Morwenna Hall