Thymidine phosphorylase

 

Thymidine phosphorylases (TP) are key enzymes in nucleoside metabolism. In the presence of inorganic phosphate, they catalyse the reversible phosphorolysis of the glycosidic bond in thymidine- and uridine-2′-deoxyribosides, with the formation of free base and deoxyribose 1-phosphate. In addition to phosphorolysis, they also catalyse the transfer of the deoxyribosyl moiety from one pyrimidine base to another. This transglycosylation reaction plays a key role in the salvage pathway of nucleoside biosyntheses, providing an alternative to the de novo purine and pyrimidine biosynthetic pathways.

There are two families of nucleoside phosphorylases, which differ remarkably in the type of quaternary structure and in polypeptide chain folding. TP belongs to the family II nucleoside phosphorylases which comprises all pyrimidine phosphorylases (PyNPs) except for uridine phosphorylase. All bacterial and mammalian pyrimidine phosphorylases of family II display a dimeric quaternary structure with subunits consisting of a small alpha domain separated by a large cleft from a larger alpha/beta domain. The biologically active form of these enzymes is a dimer.

Mammalian thymidine phosphorylases stimulate tumour growth, and their level is enhanced in tumour cells, where they are involved in angiogenesis and development of metastasis. Owing to their ability to catalyse transglycosylation, some bacterial TPs are widely used in biotechnology for the large-scale production of natural 2′-deoxy-beta-D-ribonucleosides and their analogues containing modifications in the carbohydrate and base fragments. Among these nucleosides are many biologically important compounds with anticancer and antiviral activity. Therefore, the search for compounds which are able to regulate or inhibit TP activity as well as study of the interactions of these compounds with TP is of special interest. Knowledge at the atomic level of the interaction of such compounds with TP and other enzymes of nucleoside metabolism is required for the rational design of new drugs and the engineering of new forms of the enzymes with altered selectivity.

 

Reference Protein and Structure

Sequence
P07650 UniProt (2.4.2.4) IPR018090 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1azy - STRUCTURAL AND THEORETICAL STUDIES SUGGEST DOMAIN MOVEMENT PRODUCES AN ACTIVE CONFORMATION OF THYMIDINE PHOSPHORYLASE (3.0 Å) PDBe PDBsum 1azy
Catalytic CATH Domains
3.40.1030.10 CATHdb 1.20.970.10 CATHdb (see all for 1azy)
Click To Show Structure

Enzyme Reaction (EC:2.4.2.4)

thymidine
CHEBI:17748ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
→
2-deoxy-alpha-D-ribose 1-phosphate(2-)
CHEBI:57259ChEBI
+
thymine
CHEBI:17821ChEBI
Alternative enzyme names: Animal growth regulators, blood platelet-derived endothelial cell growth factors, Blood platelet-derived endothelial cell growth factor, Deoxythymidine phosphorylase, Gliostatins, Pyrimidine deoxynucleoside phosphorylase, Pyrimidine phosphorylase, Thymidine-orthophosphate deoxyribosyltransferase, Thymidine:phosphate deoxy-D-ribosyltransferase,

Enzyme Mechanism

Introduction

First, the enzyme binds its two substrates in the crystal structure (open) geometry. The kinetic data for E. coli TP indicates that the binding is ordered sequentially, in which the phosphate ion binds first. This is followed by a conformational change of the enzyme, in which the two domains move to bring the two substrates together. In this geometry the reaction can take place in a two-step mechanism with a low activation energy. The first step is a direct nucleophilic attack of a phosphate ion oxygen atom on the C1′ atom, resulting in cleavage of the glycosidic bond. This is followed by phosphorylation of the ribose ring. This mechanism and the transition-state geometry are similar to what has been proposed for other N-glycosidic enzymes based on kinetic isotope experiments. It is likely that a final step of the mechanism involves another conformational change, after which the products can be released.

Catalytic Residues Roles

UniProt PDB* (1azy)
Arg171 Arg171A Acts as a hydrogen bond donor to stabilise the negative charge on the purine ring after the glycosidic bond is broken. In a position to stabilise dominant resonance structures of a negatively charged thymine leaving group. electrostatic stabiliser
Lys190 Lys190A Forms a hydrogen bond with the 3′-hydroxyl of the thymidine ribose moiety may be well situated to donate a proton to thymine N1. In a position to stabilise dominant resonance structures of a negatively charged thymine leaving group proton shuttle (general acid/base), electrostatic stabiliser
Lys191 Lys191A Helps activate and stabilise the phosphate substrate. electrostatic stabiliser
Lys84 Lys84A Increases the basicity of Asp83. activator, electrostatic stabiliser
Asp83 Asp83A Correctly positioned to abstract a proton from the phosphate ion (either directly or via a proton relay with Lys84). proton shuttle (general acid/base)
Ser186 Ser186A Exact role unclear. Thought to be involved in stabilising the transition states and negatively charged intermediates of the pyrimidine ring. electrostatic stabiliser
Thr123, Ser86, His85 Thr123A, Ser86A, His85A Exact role unclear, however essential to catalysis. Thought to be involved in activating and stabilising the substrate phosphate. 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

References

  1. 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.
  2. 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.
  3. Mitsiki E et al. (2009), Biochem Biophys Res Commun, 386, 666-670. Structures of native human thymidine phosphorylase and in complex with 5-iodouracil. DOI:10.1016/j.bbrc.2009.06.104. PMID:19555658.
  4. Panova NG et al. (2007), Biochemistry (Mosc), 72, 21-28. Substrate specificity of Escherichia coli thymidine phosphorylase. DOI:10.1134/s0006297907010026.
  5. Rick SW et al. (1999), Proteins, 37, 242-252. Computational studies of the domain movement and the catalytic mechanism of thymidine phosphorylase. DOI:10.1002/(sici)1097-0134(19991101)37:2<242::aid-prot9>3.0.co;2-5.
  6. 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.

Step 1.

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Catalytic Residues Roles

Residue Roles
Arg171A electrostatic stabiliser
Lys190A electrostatic stabiliser
Asp83A proton shuttle (general acid/base)
Lys84A electrostatic stabiliser, activator
Lys190A proton shuttle (general acid/base)
Lys191A electrostatic stabiliser
His85A electrostatic stabiliser
Ser86A electrostatic stabiliser
Thr123A electrostatic stabiliser
Ser186A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday