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
(2.4.2.2)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Geobacillus stearothermophilus (Bacteria)
- PDB
-
1brw
- THE CRYSTAL STRUCTURE OF PYRIMIDINE NUCLEOSIDE PHOSPHORYLASE IN A CLOSED CONFORMATION
(2.1 Å)
- Catalytic CATH Domains
-
3.40.1030.10
(see all for 1brw)
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* (1brw) | ||
| 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 |
Chemical Components
proton transfer, heterolysis, bimolecular nucleophilic addition, native state of enzyme regeneratedReferences
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Lewkowicz E et al. (2006), Curr Org Chem, 10, 1197-1215. Nucleoside Phosphorylases. DOI:10.2174/138527206777697995.
- 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.
- 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
Step 2. In a heterolysis reaction, the C-N bond between ribose and the purine is cleaved.
Download: Image, Marvin FileCatalytic 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
Step 3. Phosphate initiates a nucleophilic attack on the ribose in an addition reaction.
Download: Image, Marvin FileCatalytic 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 |
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