Author:
Timofeev Vladimir I.,Zhukhlistova Nadezhda E.,Abramchik Yuliya A.,Fateev Ilya I.,Kostromina Maria A.,Muravieva Tatiana I.,Esipov Roman S.,Kuranova Inna P.
Abstract
Purine nucleoside phosphorylases (EC 2.4.2.1; PNPs) reversibly catalyze the phosphorolytic cleavage of glycosidic bonds in purine nucleosides to generate ribose 1-phosphate and a free purine base, and are key enzymes in the salvage pathway of purine biosynthesis. They also catalyze the transfer of pentosyl groups between purine bases (the transglycosylation reaction) and are widely used for the synthesis of biologically important analogues of natural nucleosides, including a number of anticancer and antiviral drugs. Potent inhibitors of PNPs are used in chemotherapeutic applications. The detailed study of the binding of purine bases and their derivatives in the active site of PNPs is of particular interest in order to understand the mechanism of enzyme action and for the development of new enzyme inhibitors. Here, it is shown that 7-deazahypoxanthine (7DHX) is a noncompetitive inhibitor of the phosphorolysis of inosine by recombinantEscherichia coliPNP (EcPNP) with an inhibition constantKiof 0.13 mM. A crystal ofEcPNP in complex with 7DHX was obtained in microgravity by the counter-diffusion technique and the three-dimensional structure of theEcPNP–7DHX complex was solved by molecular replacement at 2.51 Å resolution using an X-ray data set collected at the SPring-8 synchrotron-radiation facility, Japan. The crystals belonged to space groupP6122, with unit-cell parametersa=b= 120.370,c= 238.971 Å, and contained three subunits of the hexameric enzyme molecule in the asymmetric unit. The 7DHX molecule was located with full occupancy in the active site of each of the three crystallographically independent enzyme subunits. The position of 7DHX overlapped with the positions occupied by purine bases in similar PNP complexes. However, the orientation of the 7DHX molecule differs from those of other bases: it is rotated by ∼180° relative to other bases. The peculiarities of the arrangement of 7DHX in theEcPNP active site are discussed.
Funder
Russian Federal Space Agency
Publisher
International Union of Crystallography (IUCr)
Subject
Condensed Matter Physics,Genetics,Biochemistry,Structural Biology,Biophysics
Cited by
2 articles.
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