Still a Long Way to Fully Understanding the Molecular Mechanism of Escherichia coli Purine Nucleoside Phosphorylase

The results of several decades of studying the catalytic mechanism of Escherichia colt 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

Crystallographic snapshots of ligand binding to hexameric purine nucleoside phosphorylase and kinetic studies give insight into the mechanism of catalysis

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

Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent

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 various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule

Biophysical studies of purine nucleoside phosphorylase from E. coli - mechanism of catalysis, relationship between protein function and its oligomeric structure

Fosforylaza nukleozydów purynowych (PNP) z Escherichia coli katalizuje odwracalną reakcję fosforolitycznego rozszczepienia nukleozydów purynowych na odpowiednią zasadę purynową i pentozo-1-fosforan. Białko jest homoheksamerem i w formie apo struktura wszystkich podjednostek jest taka sama. Dane literaturowe, dostępne w momencie rozpoczynania tej pracy, sugerowały, że strukturalnie białko jest trimerem dimerów, ponieważ miejsce aktywne każdej podjednostki w dimerze jest współtworzone przez dwa aminokwasy sąsiada z tego samego dimeru. To sugerowało również, że podstawową jednostką katalityczną białka jest dimer. Wyniki badań w roztworze, wydawały się potwierdzać tę hipotezę, ponieważ, zauważono niemichaelisowską kinetykę PNP wobec jonów fosforanowych ze współczynnikiem Hilla równym 0,5, wskazującym na silną negatywną kooperację [1]. Kilka, dostępnych w literaturze struktur krystalograficznych apo-enzymu i różnych kompleksów nie wyjaśniało mechanizmu, za pomocą którego informacja jest przekazywana między podjednostkami lub miejscami aktywnymi enzymu, ani też jaka jest rola kooperacji podjednostek w katalizie. W 2002 roku, została uzyskana struktura krystalograficzna, w której zaobserwowano zamkniętą konformację części miejsc aktywnych, po jednym w każdym dimerze, pozostałe podjednostki zachowały konformację otwartą miejsca aktywnego, jak w formie apo. Na podstawie tej struktury, został zaproponowany mechanizm katalizy, opisujący sekwencję zdarzeń w miejscu aktywnym [2], która polegałaby na zamknięciu miejsca aktywnego przy udziale Arg24. W tej konformacji miejsca aktywnego reszta Asp204, przy współpracy Arg217, może uprotonować nukleozyd, co zmienia rozkład ładunku na pierścieniu purynowym, prowadzi do destabilizacji i pęknięcia wiązania glikozydowego. Celem pracy była weryfikacja zaproponowanego mechanizmu katalizy, poprzez badania mutantów miejsca aktywnego, oraz wyjaśnienie mechanizmu i znaczenia kooperacji między podjednostkami heksamerycznego białka. Pierwszą przesłanką, że kooperacja zachodzi nie tylko wewnątrz dimeru, ale również pomiędzy nimi, były krzywe wiązania jonów fosforanowych, dla opisu których model wiązania z dwiema stałymi dysocjacji okazał się niewystarczający. Wiązanie opisywane trzema stałymi dysocjacji zostało potwierdzone przy pomocy kilku technik eksperymentalnych. Związanie pierwszego jonu fosforanowego prowadzi do powstania w białku trzech rodzajów miejsc aktywnych, jednego w konformacji zamkniętej i dwóch w konformacji otwartej, charakteryzujących się różnym powinowactwem do ligandu. Wobec tego fakt, że wiązanie nukleozydu okazało się być opisywane również trzema stałymi nie był zaskoczeniem. Istnienie trzech rodzajów miejsc wiążących zaobserwowano także w strukturach krystalograficznych. Poprzez destabilizację kontaktów pomiędzy dimerami w heksamerze, uzyskano dimery, które okazały się nie być stabilne na tyle, aby prowadzić efektywną katalizę. Mutując Asp204, Arg217 i Arg24 na alaninę i prowadząc badania kinetyczne względem naturalnych, a także imitujących stan uprotonowany, substratów, wykazano, że, jak zaproponowano w 2002 roku, zmiana konformacyjna i protonacja nukleozydu są niezbędnymi etapami katalizy. Na podstawie przeprowadzonych badań zaproponowano również mechanizm katalizy, uwzględniający kooperację pomiędzy podjednostkami białka w dimerze. W warunkach nasycenia substratami podjednostki otwarte i zamknięte w dimerach prowadzą katalizę w ścisłej współpracy. Gdy w zamkniętej podjednostce zachodzi reakcja fosforolizy, podjednostka otwarta właśnie ją zakończyła, następuje uwolnienie produktów i związanie kolejnej pary substratów, zamknięcie tej podjednostki i cykl zaczyna się od nowa. Jest to mechanizm zwany w literaturze anglojęzycznej flip-flop. Poza istnieniem trzech typów miejsc aktywnych, podczas badań białka zaobserwowano także kilka 1/2 Streszczenie innych, niespodziewanych zjawisk, które nie dawały się wyjaśnić w ramach obowiązującego dla enzymów paradygmatu, że białko jest albo aktywne, albo nieaktywne. Spadek aktywności białka przechowywanego w -80°C ma wyraźnie dwufazowy charakter. Uwolnienie stężenia białka jako parametru dopasowania modelu wiązania do krzywej miareczkowania niezmiennie zwracało niefizycznie niskie wartości. W analizie miareczkowań kalorymetrycznych, gdzie parametrem dopasowania może być także stężenie frakcji nieaktywnej białka, rozumianej jako frakcja białka niezdolnego wiązać ligand, uwolnienie tego parametru, jako parametru dopasowania zawsze zwracało wartość stężenia białka wiążącego wyższą od stężenia białka aktywnego katalitycznie, wyznaczonego na podstawie pomiarów aktywności specyficznej wobec substratu naturalnego. Wszystkie te spostrzeżenia sugerowały konieczność zbadania procesu dezaktywacji białka. Badania takie przeprowadzono i wykryto przynajmniej dwa stany przejściowe, pomiędzy białkiem w pełni aktywnym i całkowicie nieaktywnym, niezdolnym do prowadzenia katalizy i do wiązania ligandów. W pierwszym zidentyfikowanym stanie przejściowym, jeden z trzech dimerów staje się nieaktywny katalitycznie wobec naturalnych substratów, natomiast nadal jest w stanie wiązać ligandy i prowadzić fosforolizę substratów imitujących stan uprotonowany. Pozostałe dwa dimery są sprawne katalitycznie, każdy z nich zawiera podjednostkę, której miejsce aktywne przybiera konformację zamkniętą. Ta forma białka charakteryzuje się obniżoną aktywnością specyficzną w stosunku do enzymu, w którym w konformacji zamkniętej może być jedno miejsce aktywne w każdym z trzech dimerów. W kolejnym stanie przejściowym żadne z miejsc aktywnych nie może przejść do konformacji zamkniętej, zatem białko nie katalizuje fosforolizy substratów naturalnych, natomiast katalizuje fosforolizę substratów imitujących stan uprotonowany. Zaproponowano model, który wyjaśnia wszystkie zaobserwowane fakty. Pokazano, że PNP z E. coli rzeczywiście jest heksamerem, a nie trimerem niezależnie funkcjonujących dimerów, ponieważ bez "wsparcia" strukturalnego, jakie zapewniają sąsiednie podjednostki, dimer nie jest w stanie prowadzić efektywnej katalizy.Purine Nucleoside Phosphorylase (PNP) from Escherichia coli catalysis reversible reaction of phosphorolytic cleavage of purine nucleosides into corresponding purine base and pentose-1-phosphate. The protein is a hexamer, and in apo form all subunits are in the same conformation. Literature data available at the beginning of this work suggested, that structurally, the protein is in fact a trimer of dimers, because active site of particular subunit in a dimer is cocreated by two aminoacids from a neighbouring subunit of the dimer. This suggested, that the minimal catalytic unit in the protein is a dimer. Solution studies data seemed to confirm that hypothesis, as the kinetic towards phosphate ion in non-Michaelis, and is described by Hill coefficient equals 0,5, suggesting strong negative cooperation [1]. Several crystallographic structures of apoenzyme and various complexes, available in the literature, did not explain the mechanism of cooperation signal transduction between subunits or active sites, neither what is the role of co-subunits in catalysis. In 2002, the crystallographic structure was obtained, in which a closed conformation of some active sites was observed, one in each dimer, and the remaining subunits maintained the open conformation of the active site, as in the apo form. Based on this structure, a catalysis mechanism describing the sequence of events in the active site was proposed [2], which would consist of closing the active site with the participation of Arg24. In this conformation of the active site Asp204 residue, in cooperation with Arg217, can protonate nucleoside substrate. This changes the distribution of charge on the purine ring, leads to the destabilization and breakage of the glycosidic bond. The aim of the doctoral thesis was to verify the proposed mechanism of catalysis by investigating active site mutants, and to explain the mechanism and significance of cooperation between hexameric protein subunits. First indication, that cooperation does not only takes place within a dimer, but also between them, were phosphate binding curves, for which a two binding sites model was not sufficient to describe the curves properly, and three binding sites model had to be applied. Three binding site model for phosphate binding was confirmed with several experimental methods. The proposed model assumes that binding of first phosphate ion leads to formation of three kinds of binding sites, one in closed conformation and two in open conformation, but characterised with different affinity to ligands. Thus, three binding constant describing binding of nucleoside should not be surprising. Existence of three kinds of binding sites was observed also in crystal structures. Through destabilisation of contacts between dimers in hexameric ecPNP, free dimers were created, but they turned out to be not stable enough to catalyse effectively. Mechanism proposed in 2002 was confirmed by kinetic studies of active site mutants, Asp204Ala, Arg217Ala and Arg24Ala, towards natural and mimicking protonated state substrates. It was shown that conformational change and protonation of nucleoside are necessary steps of catalysis. On the basis of the conducted research, a mechanism of catalysis was proposed, taking into account the cooperation between the protein subunits in the dimer. Under conditions of substrate saturation, open and closed subunits in dimers catalyse in close cooperation. When a phosphorolysis reaction takes place in the closed subunit, the open subunit has just finished it, the products are released and the next pair of substrates are bound, the subunit is closed and the cycle begins again. This mechanism is known in the literature as flip-flop. In addition to the existence of three types of active sites, several other unexpected phenomena were observed during protein studies, that could not be explained within the framework of the paradigm that the enzyme is either active or inactive. The decrease in the activity of protein stored at -80°C has a clearly twophase nature. The optimisation of the protein concentration as a parameter of the binding model fit to the titration curve repeatedly returned non-physically low values. In the analysis of calorimetric titrations, where fraction of inactive protein, meaning a protein fraction incapable of binding ligand, can be an adjustable parameter, optimisation of this parameter as a fit parameter always returned the binding protein concentration higher than the concentration of catalytically active protein, determined from specific activity measurements vs. natural substrate. All these observations suggested the need to investigate the process of protein deactivation. Such research were performed and at least two intermediate states were detected between the fully active and completely inactive protein, unable to catalyse and to bind ligands. In the first identified transition state, one of the three dimers becomes catalytically inactive towards natural substrates, while it is still able to bind ligands and carry out phosphorolysis of substrates imitating the protonated state. The other two dimers are catalytically functional and each of them contains a subunit whose active site adopts a closed conformation. This form of the protein is characterized by a reduced specific activity compared to an enzyme, in which one closed site can be present in each of three dimers. In the next transitional state, none of the active sites can adopt a closed conformation, therefore, the protein does not catalyse the phosphorolysis of natural substrates, but it still catalyses phosphorolysis of substrates mimicking protonated state. A model that explains all observed facts was proposed. It was shown, that PNP from E. coli is indeed a hexamer, and not a trimer of independently functioning dimers, because without structural "support" provided by neighbouring subunits, the dimer is unable to catalyse effectively

New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined HD Exchange Approach and Molecular Dynamics Simulations.

The biologically active form of purine nucleoside phosphorylase (PNP) from Escherichia coli (EC 2.4.2.1) is a homohexamer unit, assembled as a trimer of dimers. Upon binding of phosphate, neighboring monomers adopt different active site conformations, described as open and closed. To get insight into the functions of the two distinctive active site conformations, virtually inactive Arg24Ala mutant is complexed with phosphate; all active sites are found to be in the open conformation. To understand how the sites of neighboring monomers communicate with each other, we have combined H/D exchange (H/DX) experiments with molecular dynamics (MD) simulations. Both methods point to the mobility of the enzyme, associated with a few flexible regions situated at the surface and within the dimer interface. Although H/DX provides an average extent of deuterium uptake for all six hexamer active sites, it was able to indicate the dynamic mechanism of cross-talk between monomers, allostery. Using this technique, it was found that phosphate binding to the wild type (WT) causes arrest of the molecular motion in backbone fragments that are flexible in a ligand-free state. This was not the case for the Arg24Ala mutant. Upon nucleoside substrate/inhibitor binding, some release of the phosphate-induced arrest is observed for the WT, whereas the opposite effects occur for the Arg24Ala mutant. MD simulations confirmed that phosphate is bound tightly in the closed active sites of the WT; conversely, in the open conformation of the active site of the WT phosphate is bound loosely moving towards the exit of the active site. In Arg24Ala mutant binary complex Pi is bound loosely, too

The pursuit of new alternative ways to eradicate Helicobacter pylori continues: Detailed characterization of interactions in the adenylosuccinate synthetase active site

Purine nucleotide synthesis is realised only through the salvage pathway in pathogenic bacterium Helicobacter pylori. Therefore, the enzymes of this pathway, among them also the adenylosuccinate synthetase (AdSS), present potential new drug targets. This paper describes characterization of His6-tagged AdSS from H. pylori. Thorough analysis of 3D-structures of fully ligated AdSS (in a complex with guanosine diphosphate, 6-phosphoryl-inosine monophosphate, hadacidin and Mg2+) and AdSS in a complex with inosine monophosphate (IMP) only, enabled identification of active site interactions crucial for ligand binding and enzyme activity. Combination of experimental and molecular dynamics (MD) simulations data, particularly emphasized the importance of hydrogen bond Arg135-IMP for enzyme dimerization and active site formation. The synergistic effect of substrates (IMP and guanosine triphosphate) binding was suggested by MD simulations. Several flexible elements of the structure (loops) are stabilized by the presence of IMP alone, however loops comprising residues 287–293 and 40–44 occupy different positions in two solved H. pylori AdSS structures. MD simulations discovered the hydrogen bond network that stabilizes the closed conformation of the residues 40–50 loop, only in the presence of IMP. Presented findings provide a solid basis for the design of new AdSS inhibitors as potential drugs against H. pylori

1.45\AA resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state

Low molecular mass purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) are homotrimeric enzymes that are tightly inhibited by immucillins. Due to the positive charge on the ribose like part (iminoribitol moiety) and protonation of the N7 atom of the purine ring, immucillins are believed to act as transition state analogues. Over a wide range of concentrations, immucillins bind with strong negative cooperativity to PNPs, so that only every third binding site of the enzyme is occupied (third-of-the-sites binding). 9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) shares with immucillins the protonation of the N7, but not the positive charge on the ribose like part of the molecule. We have previously shown that DFPP-DG interacts with PNPs with subnanomolar inhibition constant. Here, we report additional biochemical experiments to demonstrate that the inhibitor can be bound with the same K(d) ( approximately 190pM) to all three substrate binding sites of the trimeric PNP, and a crystal structure of PNP in complex with DFPP-DG at 1.45A resolution, the highest resolution published for PNPs so far. The crystals contain the full PNP homotrimer in the asymmetric unit. DFPP-DG molecules are bound in superimposable manner and with full occupancies to all three PNP subunits. Thus the postulated third-of-the-sites binding of immucillins should be rather attribute to the second feature of the transition state, ribooxocarbenium ion character of the ligand or to the coexistence of both features characteristic for the transition state. The DFPP-DG/PNP complex structure confirms the earlier observations, that the loop from Pro57 to Gly66 covering the phosphate-binding site cannot be stabilized by phosphonate analogues. The loop from Glu250 to Gln266 covering the base-binding site is organized by the interactions of Asn243 with the Hoogsteen edge of the purine base of analogues bearing one feature of the postulated transition state (protonated N7 position)