Dataset Information

Crystal structure of Escherichia coli purine nucleoside phosphorylase in complex with 7-deazahypoxanthine.

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 recombinant Escherichia coli PNP (EcPNP) with an inhibition constant Ki of 0.13?mM. A crystal of EcPNP in complex with 7DHX was obtained in microgravity by the counter-diffusion technique and the three-dimensional structure of the EcPNP-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 group P6122, with unit-cell parameters a = 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 the EcPNP active site are discussed.

SUBMITTER: Timofeev VI

PROVIDER: S-EPMC5987744 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Crystal structure of Escherichia coli purine nucleoside phosphorylase in complex with 7-deazahypoxanthine.

Timofeev Vladimir I VI Zhukhlistova Nadezhda E NE Abramchik Yuliya A YA Fateev Ilya I II Kostromina Maria A MA Muravieva Tatiana I TI Esipov Roman S RS Kuranova Inna P IP

Acta crystallographica. Section F, Structural biology communications 20180523 Pt 6

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 ...[more]

PMID: 29870020

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Project description:Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction.

Dataset Information

Crystal structure of Escherichia coli purine nucleoside phosphorylase in complex with 7-deazahypoxanthine.

Publications

Crystal structure of Escherichia coli purine nucleoside phosphorylase in complex with 7-deazahypoxanthine.

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