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Acta Crystallogr D Biol Crystallogr. 2014 Apr;70(Pt 4):1155-65. doi: 10.1107/S1399004714001904. Epub 2014 Mar 21.

3'-Azidothymidine in the active site of Escherichia coli thymidine phosphorylase: the peculiarity of the binding on the basis of X-ray study.

Author information

  • 1X-ray Analysis Methods and Synchrotron Radiation, Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninsky Prospect 59, Moscow, 119333, Russian Federation.
  • 2Laboratory of Biotechnology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russian Federation.

Abstract

The structural study of complexes of thymidine phosphorylase (TP) with nucleoside analogues which inhibit its activity is of special interest because many of these compounds are used as chemotherapeutic agents. Determination of kinetic parameters showed that 3'-azido-3'-deoxythymidine (3'-azidothymidine; AZT), which is widely used for the treatment of human immunodeficiency virus, is a reversible noncompetitive inhibitor of Escherichia coli thymidine phosphorylase (TP). The three-dimensional structure of E. coli TP complexed with AZT was solved by the molecular-replacement method and was refined at 1.52 Å resolution. Crystals for X-ray study were grown in microgravity by the counter-diffusion technique from a solution of the protein in phosphate buffer with ammonium sulfate as a precipitant. The AZT molecule was located with full occupancy in the electron-density maps in the nucleoside-binding pocket of TP, whereas the phosphate-binding pocket of the enzyme was occupied by phosphate (or sulfate) ion. The structure of the active-site cavity and conformational changes of the enzyme upon AZT binding are described in detail. It is found that the position of AZT differs remarkably from the positions of the pyrimidine bases and nucleoside analogues in other known complexes of pyrimidine phosphorylases, but coincides well with the position of 2'-fluoro-3'-azido-2',3'-dideoxyuridine (N3FddU) in the recently investigated complex of E. coli TP with this ligand (Timofeev et al., 2013). The peculiarities of the arrangement of N3FddU and 3'-azidothymidine in the nucleoside binding pocket of TP and correlations between the arrangement and inhibitory properties of these compounds are discussed.

KEYWORDS:

3′-azidothymidine; active site; thymidine phosphorylase; zidovudine

PMID:
24699659
[PubMed - in process]
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