Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution (original) (raw)
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Acta Crystallographica Section F: Structural Biology Communications, 2018
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 K i of 0.13 mM. A crystal of EcPNP in complex with 7DHX was obtained in microgravity by the counter-diffusion technique and the threedimensional 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 P6 1 22, 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.
FEBS Letters, 2012
Purine nucleoside phosphorylase (PNP) from Escherichia coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. The first crystal structure of the ternary complex of this enzyme (with a phosphate ion and formycin A), which is biased by neither the presence of an inhibitor nor sulfate as a precipitant, is presented. The structure reveals, in some active sites, an unexpected and never before observed binding site for phosphate and exhibits a stoichiometry of two phosphate molecules per enzyme subunit. Moreover, in these active sites, the phosphate and nucleoside molecules are found not to be in direct contact. Rather, they are bridged by three water molecules that occupy the ''standard'' phosphate binding site.
FEBS Letters, 2012
Purine nucleoside phosphorylase (PNP) from Escherichia coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. The first crystal structure of the ternary complex of this enzyme (with a phosphate ion and formycin A), which is biased by neither the presence of an inhibitor nor sulfate as a precipitant, is presented. The structure reveals, in some active sites, an unexpected and never before observed binding site for phosphate and exhibits a stoichiometry of two phosphate molecules per enzyme subunit. Moreover, in these active sites, the phosphate and nucleoside molecules are found not to be in direct contact. Rather, they are bridged by three water molecules that occupy the ''standard'' phosphate binding site. Structured summary of protein interactions: PNP and PNP bind by x-ray crystallography (View interaction) .pl (A. Bzowska), marija.luic@irb.hr (M. Luić ). FEBS Letters 586 (2012) 967-971 j o u r n a l h o m e p a g e : w w w . F E B S L e t t e r s . o r g
Croatica Chemica Acta, 2013
The results of several decades of studying the catalytic mechanism of Escherichia coli purine nucleoside phosphorylases (PNP) by solution studies and crystal structure determinations are presented. Potentially PNPs can be used for enzyme-activating prodrug gene therapy against solid tumours because of the differences in specificity between human and E. coli PNPs. Biologically active form of PNP from E. coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. Two conformations of the active site are possible after substrate(s) binding: open and closed. A series of determined 3D-structures of PNP binary and ternary complexes facilitated the prediction of the main steps in the catalytic mechanism. For their validation the active site mutants: Arg24Ala, Asp204Ala, Arg217Ala, Asp204Asn and double mutant Asp204Ala/Arg217Ala were prepared. The activity tests confirm that catalysis involves protonation of the purine base at position N7 and give better insight into the cooperativity between subunits in this oligomeric enzyme.
Scientific Reports
Purine nucleoside phosphorylase (PNP) catalyses the cleavage of the glycosidic bond of purine nucleosides using phosphate instead of water as a second substrate. PNP from Escherichia coli is a homohexamer, build as a trimer of dimers, and each subunit can be in two conformations, open or closed. This conformational change is induced by the presence of phosphate substrate, and very likely a required step for the catalysis. Closing one active site strongly affects the others, by a yet unclear mechanism and order of events. Kinetic and ligand binding studies show strong negative cooperativity between subunits. Here, for the first time, we managed to monitor the sequence of nucleoside binding to individual subunits in the crystal structures of the wild-type enzyme, showing that first the closed sites, not the open ones, are occupied by the nucleoside. However, two mutations within the active site, Asp204Ala/Arg217Ala, are enough not only to significantly reduce the effectiveness of the enzyme, but also reverse the sequence of the nucleoside binding. In the mutant the open sites, neighbours in a dimer of those in the closed conformation, are occupied as first. This demonstrates how important for the effective catalysis of Escherichia coli PNP is proper subunit cooperation. Purine nucleoside phosphorylase (PNP, purine nucleoside orthophosphate ribosyl transferase, EC 2.4.2.1) has a crucial role in the purine salvage pathway 1. It catalyses the reversible phosphorolytic cleavage of the glycosidic bond of purine (2′-deoxy) nucleosides, generating the corresponding free base and (2′-deoxy) ribose 1-phosphate 1,2. The biologically active form of this enzyme, with only one known exception-PNP from Thermus thermophilus HB27 3 , is always oligomeric: homotrimers are characteristic mostly for mammals, while homohexamers are typical for most bacteria. The arrangement of subunits in the structure of a PNP hexamer is such that two of them donate two amino acids (His4 and Arg43 in E. coli) to each other, thus completing the active site of its neighbour, effectively forming a dimer which possesses an approximate 2-fold symmetry. Three such dimers are then arranged by a 3-fold symmetry axis to form a hexamer. The molecular mechanism by which this enzyme accomplishes its biological function is particularly intricate. In addition to the phosphorolysis being a two-substrate and two-product reversible reaction, with communication between monomers forming a dimer 1 , the mechanism is further complicated by the higher order communication, i.e. an allosteric cross-talk between dimers forming the hexamer 4,5. The way in which this allosteric communication is achieved is still unclear, but the complexity is reflected in an ever increasing number of different arrangements of active site conformations between monomers in the available crystal structures of hexameric PNPs 6. The catalytic mechanism of PNP, especially that of E. coli PNP, has been in the focus of our study for many years 2,5,7-16. It has been firmly established 5,14 that the initial step in the catalysis involves breaking a single α-helix
FEBS Journal, 2014
Although many enzymes are homooligomers composed of tightly bound subunits, it is often the case that smaller assemblies of such subunits, or even individual monomers, seem to have all the structural features necessary to independently conduct catalysis. In this study, we investigated the reasons justifying the necessity for the hexameric form of Escherichia coli purine nucleoside phosphorylasea homohexamer composed of three linked dimerssince it appears that the dimer is the smallest unit capable of catalyzing the reaction, according to the currently accepted mechanism. Molecular modelling was employed to probe mutations at the dimer-dimer interface that would result in a dimeric enzyme form. In this way, both in silico and in vitro, the hexamer was successfully transformed into dimers. However, modelling and solution studies show that, when isolated, dimers cannot maintain the appropriate three-dimensional structure, including the geometry of the active site and the position of the catalytically important amino acids. Analytical ultracentrifugation proves that E. coli purine nucleoside phosphorylase dimeric mutants tend to dissociate into monomers with dissociation constants of 20-80 lM. Consistently, the catalytic activity of these mutants is negligible, at least 6 orders of magnitude smaller than for the wild-type enzyme. We conclude that the hexameric architecture of E. coli purine nucleoside phosphorylase is necessary to provide stabilization of the proper three-dimensional structure of the dimeric assembly, and therefore this enzyme is the obligate (obligatory) hexamer.
Protein Science, 2006
Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150 6 10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20°C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 mg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s 0 20,w ¼ 7.7 6 0.3 and 8.3 6 0.4 S, respectively. The molecular mass of the sedimenting particle (146 6 30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.
Croatica Chemica Acta
It is generaly believed that enzymes retain most of their functionality in the crystal form due to the large solvent content of protein crystals. This is facilitated by the fact that their natural environment in solution is not too far from the one found in the crystal form. Nevertheless, if the nature of the enzyme is such to require conformational changes, overcoming of the crystal packing constraints may prove to be too difficult. Such conformational change is present in one class of enzymes (purine nucleoside phosphorylases), that is the subject of our scientific interest for many years. The influence of crystal symmetry and crystal packing on the conformation of the active sites in the case of homohexameric purine nucleoside phosphorylases is presented and analysed.
Scientific Reports
E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein–ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing va...