Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme.

Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.

Interactions of 2,6-substituted purines with purine nucleoside phosphorylase from Helicobacter pylori in solution and in the crystal, and the effects of these compounds on cell cultures of this bacterium.

Helicobacter pylori represents a global health threat with around 50% of the world population infected. Due to the increasing number of antibiotic-resistant strains, new strategies for eradication of H. pylori are needed. In this study, we suggest purine nucleoside phosphorylase (PNP) as a possible new drug target, by characterising its interactions with 2- and/or 6-substituted purines as well as the effect of these compounds on bacterial growth. Inhibition constants are in the micromolar range, the lowest being that of 6-benzylthio-2-chloropurine. This compound also inhibits H. pylori 26695 growth at the lowest concentration. X-ray structures of the complexes of PNP with the investigated compounds allowed the identification of interactions of inhibitors in the enzyme's base-binding site and the suggestion of structures that could bind to the enzyme more tightly. Our findings prove the potential of PNP inhibitors in the design of drugs against H. pylori.

A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by H. pylori.

Due to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium's recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori - two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori. KEY POINTS: • A method for assessing H. pylori cells penetration by drug candidates is described. • Three alternative detection assays that complement each other can be used. • The method may be adapted for other bacteria as well.

Chromophore of an Enhanced Green Fluorescent Protein Can Play a Photoprotective Role Due to Photobleaching.

Under stress conditions, elevated levels of cellular reactive oxygen species (ROS) may impair crucial cellular structures. To counteract the resulting oxidative damage, living cells are equipped with several defense mechanisms, including photoprotective functions of specific proteins. Here, we discuss the plausible ROS scavenging mechanisms by the enhanced green fluorescent protein, EGFP. To check if this protein could fulfill a photoprotective function, we employed electron spin resonance (ESR) in combination with spin-trapping. Two organic photosensitizers, rose bengal and methylene blue, as well as an inorganic photocatalyst, nano-TiO2, were used to photogenerate ROS. Spin-traps, TMP-OH and DMPO, and a nitroxide radical, TEMPOL, served as molecular targets for ROS. Our results show that EGFP quenches various forms of ROS, including superoxide radicals and singlet oxygen. Compared to the three proteins PNP, papain, and BSA, EGFP revealed high ROS quenching ability, which suggests its photoprotective role in living systems. Damage to the EGFP chromophore was also observed under strong photo-oxidative conditions. This study contributes to the discussion on the protective function of fluorescent proteins homologous to the green fluorescent protein (GFP). It also draws attention to the possible interactions of GFP-like proteins with ROS in systems where such proteins are used as biological markers.

Molecular Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies.

Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.

Tricyclic Nucleobase Analogs and Their Ribosides as Substrates and Inhibitors of Purine-Nucleoside Phosphorylases III. Aminopurine Derivatives.

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9- -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

Tri-Cyclic Nucleobase Analogs and their Ribosides as Substrates of Purine-Nucleoside Phosphorylases. II Guanine and Isoguanine Derivatives.

Etheno-derivatives of guanine, O6-methylguanine, and isoguanine were prepared and purified using standard methods. The title compounds were examined as potential substrates of purine-nucleoside phosphorylases from various sources in the reverse (synthetic) pathway. It was found that 1,N2-etheno-guanine and 1,N6-etheno-isoguanine are excellent substrates for purine-nucleoside phosphorylase (PNP) from E. coli, while O6-methyl-N2,3-etheno-guanine exhibited moderate activity vs. this enzyme. The latter two compounds displayed intense fluorescence in neutral aqueous medium, and so did the corresponding ribosylation products. By contrast, PNP from calf spleens exhibited only modest activity towards 1,N6-etheno-isoguanine; the remaining compounds were not ribosylated by this enzyme. The enzymatic ribosylation of 1,N6-etheno-isoguanine using two forms of calf PNP (wild type and N243D) and E. coli PNP (wild type and D204N) gave three different products, which were identified on the basis of NMR analysis and comparison with the product of the isoguanosine reaction with chloroacetic aldehyde, which gave an essentially single compound, identified unequivocally as N9-riboside. With the wild-type E. coli enzyme as a catalyst, N9--d- and N7--d-ribosides are obtained in proportion ~1:3, while calf PNP produced another riboside, tentatively identified as N6--d-riboside. The potential application of various forms of PNP for synthesis of the tri-cyclic nucleoside analogs is discussed.

Part-of-the-sites binding and reactivity in the homooligomeric enzymes - facts and artifacts.

For a number of enzymes composed of several subunits with the same amino acid sequence, it was documented, or suggested, that binding of a ligand, or catalysis, is carried out by a single subunit. This phenomenon may be the result of a pre-existent asymmetry of subunits or a limiting case of the negative cooperativity, and is sometimes called "half-of-the-sites binding (or reactivity)" for dimers and could be called "part-of-the-sites binding (or reactivity)" for higher oligomers. In this article, we discuss molecular mechanisms that may result in "part-of-the-sites binding (and reactivity)", offer possible explanations why it may have a beneficial role in enzyme function, and point to experimental problems in documenting this behaviour. We describe some cases, for which such a mechanism was first reported and later disproved. We also give several examples of enzymes, for which this mechanism seems to be well documented, and profitable. A majority of enzymes identified in this study as half-of-the-sites binding (or reactive) use it in the flip-flop version, in which "half-of-the-sites" refers to a particular moment in time. In general, the various variants of the mechanism seems to be employed often by oligomeric enzymes for allosteric regulation to enhance the efficiency of enzymatic reactions in many key metabolic pathways.

In the quest for new targets for pathogen eradication: the adenylosuccinate synthetase from the bacterium Helicobacter pylori.

Adenylosuccinate synthetase (AdSS) is an enzyme at regulatory point of purine metabolism. In pathogenic organisms which utilise only the purine salvage pathway, AdSS asserts itself as a promising drug target. One of these organisms is Helicobacter pylori, a wide-spread human pathogen involved in the development of many diseases. The rate of H. pylori antibiotic resistance is on the increase, making the quest for new drugs against this pathogen more important than ever. In this context, we describe here the properties of H. pylori AdSS. This enzyme exists in a dimeric active form independently of the presence of its ligands. Its narrow stability range and pH-neutral optimal working conditions reflect the bacterium's high level of adaptation to its living environment. Efficient inhibition of H. pylori AdSS with hadacidin and adenylosuccinate gives hope of finding novel drugs that aim at eradicating this dangerous pathogen.

Site-Selective Ribosylation of Fluorescent Nucleobase Analogs Using Purine-Nucleoside Phosphorylase as a Catalyst: Effects of Point Mutations.

Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-ß-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-ß-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.

[Purine and pyrimidine nucleoside phosphorylases - remarkable enzymes still not fully understood]. / Nukleozydowe fosforylazy purynowe i pirymidynowe - niezwykle enzymy ciagle nie do konca rozszyfrowane.

Purine and pyrimidine nucleoside phosphorylases catalyze the reversible phosphorolytic cleavage of the glycosidic bond of purine and pyrimidine nucleosides, and are key enzymes of the nucleoside salvage pathway. This metabolic route is the less costly alternative to the de novo synthesis of nucleosides and nucleotides, supplying cells with these important building blocks. Interest in nucleoside phosphorylases is not only due to their important role in metabolism of nucleosides and nucleotides, but also due to the potential medical use of the enzymes (all phosphorylases in activating prodrugs - nucleoside and nucleic base analogs, high-molecular mass purine nucleoside phosphorylases in gene therapy of some solid tumors) and their inhibitors (as selective immunosuppressive, anticancer and antiparasitic agents, and preventing inactivation of other nucleoside drugs). Phosphorylases are also convenient tools for efficient enzymatic synthesis of otherwise inaccessible nucleoside analogues. In this paper the contribution of Professor David Shugar and some of his colleagues and coworkers in studies of these remarkable enzymes carried out over nearly 40 years is discussed on the background of global research in this field.

Purine nucleoside phosphorylase activity decline is linked to the decay of the trimeric form of the enzyme.

Homotrimeric mammalian purine nucleoside phosphorylase (PNP) plays a key role in the nucleoside and nucleotide metabolic salvage pathway. Each monomer in the active PNP trimer is composed of a central ß-sheet flanked by several α-helices. We investigated the stability of calf PNP using analytical ultracentrifugation, differential scanning calorimetry, circular dichroism, and UV absorption spectroscopy. The results demonstrate that the activity decline (due to protein aging after isolation from cells) of wild type PNP and its two mutants with point mutations in the region of monomer-monomer interface, is accompanied by a decrease of the population of the trimeric enzyme and an increase of the population of its aggregated forms. The data do not indicate a significant population of either folded or unfolded PNP monomers. The enzyme with specific activity lower than the maximal shows a decrease of the helical structure, which can make it prone to aggregation. The presence of phosphate stabilizes the enzyme but leads to a more pronounced aggregation above the melting temperature. These results suggest that the biological role of packing of the PNP monomers into a trimeric structure is to provide the stability of the enzyme since the monomers are not stable in solution.

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.

Trimeric purine nucleoside phosphorylase: exploring postulated one-third-of-the-sites binding in the transition state.

Transition-state analogue inhibitors, immucillins, were reported to bind to trimeric purine nucleoside phosphorylase (PNP) with the stoichiometry of one molecule per enzyme trimer [Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L. Biochem. 1998, 37, 8615]. In attempts to observe and better understand the nature of this phenomenon we have conducted calorimetric titrations of the recombinant calf PNP complexed with immucillin H. However, by striking contrast to the earlier reports, we have not observed negative cooperativity and we got the stoichiometry of three immucillin molecules per enzyme trimer. Similar results were obtained from fluorimetric titrations, and for other inhibitors bearing features of the transition state. However, we observed apparent cooperativity between enzyme subunits and apparent lower stoichiometry when we used the recombinant enzyme not fully purified from hypoxanthine, which is moped from Escherichia coli cells. Results presented here prove that one-third-of-the-sites binding does not occur for trimeric PNP, and give the highly probable explanation why previous experiments were interpreted in terms of this phenomenon.

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.

Hierarchical approach for the rational construction of helix-containing nanofibrils using α,ß-peptides.

The rational design of novel self-assembled nanomaterials based on peptides remains a great challenge in modern chemistry. A hierarchical approach for the construction of nanofibrils based on α,ß-peptide foldamers is proposed. The incorporation of a helix-promoting trans-(1S,2S)-2-aminocyclopentanecarboxylic acid residue in the outer positions of the model coiled-coil peptide led to its increased conformational stability, which was established consistently by the results of CD, NMR and FT-IR spectroscopy. The designed oligomerization state in the solution of the studied peptides was confirmed using analytical ultracentrifugation. Moreover, the cyclopentane side chain allowed additional interactions between coiled-coil-like structures to direct the self-assembly process towards the formation of well-defined nanofibrils, as observed using AFM and TEM techniques.

Overexpressed proteins may act as mops removing their ligands from the host cells: a case study of calf PNP.

Calf purine nucleoside phosphorylase (PNP) was overexpressed in Escherichia coli. The basic kinetic parameters of recombinant PNP were found to be similar to the values published previously for non-recombinant PNP from calf spleen. However, upon titration of the recombinant enzyme with the tight-binding multisubstrate analogue inhibitor DFPP-DG, endothermic as well as exothermic signals were obtained. This was not the case for PNP isolated from calf spleen for which only the endothermic process was observed. Further calorimetric titrations of the recombinant and non-recombinant enzyme with its potent and moderate ligands, and studied involving partial inactivation of the enzyme, lead to the conclusion that a part of the recombinant enzyme forms a complex with its product, hypoxanthine, although hypoxanthine was not present at any purification stage except for its natural occurrence in E. coli cells. Binding of hypoxanthine is accompanied with a large negative change of the free enthalpy, and therefore the replacement of this compound by DFPP-DG yields positive heat signal. Our data obtained with calf PNP indicate that similar processes--moping of ligands from the host cells--may take place in the case of other proteins with high overexpression yield.

1.45 A 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).

Structural-based design and synthesis of novel 9-deazaguanine derivatives having a phosphate mimic as multi-substrate analogue inhibitors for mammalian PNPs.

9-(5',5'-Difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) was designed as a multi-substrate analogue inhibitor against purine nucleoside phosphorylase (PNP) on the basis of X-ray crystallographic data obtained for a binary complex of 9-(5',5'-difluoro-5'-phosphonopentyl)guanine (DFPP-G) with calf-spleen PNP. DFPP-DG and its analogous compounds were synthesized by the Sonogashira coupling reaction between a 9-deaza-9-iodoguanine derivative and omega-alkynyldifluoromethylene phosphonates as a key reaction. The experimental details focused on the synthetic chemistry along with some insights into the physical and biological properties of newly synthesized DFPP-DG derivatives are disclosed.

Heterodimerizing helices as tools for nanoscale control of the organization of protein-protein and protein-quantum dots.

In this study, we tested the possibility of creating complexes of two proteins by fusing them with heterodimerizing helices. We used the fluorescent proteins GFP and mCHERRY expressed with a His-tag as our model system. We added heterodimer-forming sequences at the C- or N- termini of the proteins, opposite to the His-tag position. Heterodimerization was tested for both helices at the C-terminus or at the N- terminus and C-terminus. We observed complex formation with a nanomolar dissociation constant in both cases that was higher by one order of magnitude than the Kds measured for helices alone. The binding of two C-terminal helices was accompanied by an increased enthalpy change. The binding between helices could be stabilized by introducing an additional turn of the helix with cysteine, which was capable of forming disulphide bridges. Covalently linked proteins were obtained using this strategy and observed using fluorescence cross-correlation spectroscopy. Finally, we demonstrated the formation of complexes of protein dimers and quantum dots.