Pyrimidine-nucleoside phosphorylase

 

Pyrimidine-nucleoside phosphorylase (Py-NP) catalyses the reversible phosphorylation of both uridine and thymidine in the nucleotide synthesis salvage pathway. It is found in all lower organisms whilst in higher organisms such phosphorylases are specific to those individual nucleosides. Nevertheless, Py-NPs share 40% sequence similarity with thymidine specific phosphorylase seen in higher organisms, a prominent drug target in treating breast cancer.

 

Reference Protein and Structure

Sequence
P77836 UniProt (2.4.2.2) IPR018090 (Sequence Homologues) (PDB Homologues)
Biological species
Geobacillus stearothermophilus (Bacteria) Uniprot
PDB
1brw - THE CRYSTAL STRUCTURE OF PYRIMIDINE NUCLEOSIDE PHOSPHORYLASE IN A CLOSED CONFORMATION (2.1 Å) PDBe PDBsum 1brw
Catalytic CATH Domains
3.40.1030.10 CATHdb (see all for 1brw)
Click To Show Structure

Enzyme Reaction (EC:2.4.2.2)

uridine
CHEBI:16704ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
uracil
CHEBI:17568ChEBI
+
alpha-D-ribose 1-phosphate(2-)
CHEBI:57720ChEBI
Alternative enzyme names: Py-NPase, Pdp (gene name),

Enzyme Mechanism

Introduction

A probable mechanism of pyrimidine phosphorylation is similar to the Sn1 type reaction proposed for purine nucleoside phosphorylase - the polarisation of the N1-C1' glycosidic bond. This polarisation is encourages by three major factors: the nucleoside could bind in high energy conformation which would strain the glycosidic bond; the flow of electrons from the glycosidic bond to the pyrimidine ring could be readily stabilised by positive charges of Arg168 and Lys187; and the resulting partial positive charge at C1' could be stabilised by the formation of an oxocarbenium ion at O4', which in turn would be stabilised by the negative charge on O4 of the phosphate ion. Once the partial positive charge has built up at the C1' position of the glycosidic bond, the O4 of the phosphate ion attacks at the C1' position of the pyridimine nucleoside resulting in a pyrimidine base and alpha-D-ribose1-phosphate as products. The cleaved pyrimidine can then be protonated by His82.

Catalytic Residues Roles

UniProt PDB*
Asp161 Asp161A Stabilises the positively charged Lys187. electrostatic stabiliser
His82 His82A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Arg168, Ser183, Lys187 Arg168A, Ser183A, Lys187A Stabilises the substrate pyrimidine and its intermediates in the active site. hydrogen bond donor, 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

proton transfer, heterolysis, bimolecular nucleophilic addition, native state of enzyme regenerated

References

  1. Pugmire MJ et al. (1998), Structure, 6, 1467-1479. The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation. DOI:10.1016/s0969-2126(98)00145-2. PMID:9817849.
  2. Balaev VV et al. (2018), Acta Crystallogr F Struct Biol Commun, 74, 193-197. Crystal structure of pyrimidine-nucleoside phosphorylase from Bacillus subtilis in complex with imidazole and sulfate. DOI:10.1107/S2053230X18002935. PMID:29633966.
  3. Balaev VV et al. (2016), Acta Crystallogr F Struct Biol Commun, 72, 224-233. Structural investigation of the thymidine phosphorylase from Salmonella typhimurium in the unliganded state and its complexes with thymidine and uridine. DOI:10.1107/s2053230x1600162x. PMID:26919527.
  4. Elamin YY et al. (2016), Cancer Microenviron, 9, 33-43. Thymidine Phosphorylase in Cancer; Enemy or Friend? DOI:10.1007/s12307-015-0173-y. PMID:26298314.
  5. Timofeev V et al. (2014), Acta Crystallogr D Biol Crystallogr, 70, 1155-1165. 3′-Azidothymidine in the active site ofEscherichia colithymidine phosphorylase: the peculiarity of the binding on the basis of X-ray study. DOI:10.1107/s1399004714001904. PMID:24699659.
  6. Gao XF et al. (2006), J Struct Biol, 154, 20-26. Role of each residue in catalysis in the active site of pyrimidine nucleoside phosphorylase from Bacillus subtilis: A hybrid QM/MM study. DOI:10.1016/j.jsb.2005.11.014. PMID:16469506.
  7. Lewkowicz E et al. (2006), Curr Org Chem, 10, 1197-1215. Nucleoside Phosphorylases. DOI:10.2174/138527206777697995.
  8. Caradoc-Davies TT et al. (2004), J Mol Biol, 337, 337-354. Crystal Structures of Escherichia coli Uridine Phosphorylase in Two Native and Three Complexed Forms Reveal Basis of Substrate Specificity, Induced Conformational Changes and Influence of Potassium. DOI:10.1016/j.jmb.2004.01.039. PMID:15003451.
  9. Pugmire MJ et al. (1998), J Mol Biol, 281, 285-299. Structural and theoretical studies suggest domain movement produces an active conformation of thymidine phosphorylase. DOI:10.1006/jmbi.1998.1941. PMID:9698549.

Catalytic Residues Roles

Residue Roles
His82A hydrogen bond acceptor
Asp161A electrostatic stabiliser
His82A proton acceptor

Chemical Components

proton transfer

Catalytic Residues Roles

Residue Roles
Arg168A hydrogen bond donor
His82A hydrogen bond donor
Ser183A hydrogen bond acceptor
Lys187A hydrogen bond donor
Asp161A electrostatic stabiliser
Arg168A electrostatic stabiliser
Ser183A electrostatic stabiliser
Lys187A electrostatic stabiliser
His82A electrostatic stabiliser

Chemical Components

heterolysis

Catalytic Residues Roles

Residue Roles
Arg168A hydrogen bond donor, electrostatic stabiliser
His82A hydrogen bond donor, electrostatic stabiliser
Ser183A hydrogen bond acceptor, electrostatic stabiliser
Lys187A hydrogen bond donor, electrostatic stabiliser
Asp161A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition

Catalytic Residues Roles

Residue Roles
Arg168A hydrogen bond donor, electrostatic stabiliser
His82A hydrogen bond donor, electrostatic stabiliser
Ser183A hydrogen bond acceptor, electrostatic stabiliser
Lys187A hydrogen bond donor, electrostatic stabiliser
Asp161A electrostatic stabiliser
His82A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Gail J. Bartlett, Atlanta Cook, Craig Porter, Morwenna Hall