The Role of Phosphate Binding in Purine Nucleoside Phosphorylase of Helicobacter pylori

Purine nucleoside phosphorylase (PNP) is an essential enzyme in the purine salvage pathway of Helicobacter pylori. Since H. pylori lacks the ability to synthesize purine nucleosides de novo, inhibition of this enzyme could stop the growth of this bacterium. However, for the design of successful inhibitors the details of the mechanism of this enzyme should be fully understood. PNPs catalyze cleavage of the glycosidic bond of purine nucleosides, and phosphate is one of the substrates. It is thought that binding of phosphate induces the conformational change as a necessary initial step in the catalysis. This conformational change is manifested in closing of either one of the six active sites in the homohexameric PNPs. It is unclear whether the binding of phosphate is sufficient or just a necessary condition for the closing of the active site. In this paper we conducted an experiment to check this by soaking the crystals of the apo form of the enzyme in increasing concentrations of phosphate. This work is licensed under a Creative Commons Attribution 4.0 International License

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

Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori

first_pagesettingsOrder Article Reprints Open AccessArticle Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori by Marija Zora Mišković 1,†,Marta Wojtyś 2,†,Maria Winiewska-Szajewska 2,3ORCID,Beata Wielgus-Kutrowska 2ORCID,Marija Matković 4,Darija Domazet Jurašin 5ORCID,Zoran Štefanić 5ORCID,Agnieszka Bzowska 2,*ORCID andIvana Leščić Ašler 5,*ORCID 1 Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia 2 Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland 4 Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia 5 Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia * Authors to whom correspondence should be addressed. † These authors contributed equally to this work. Int. J. Mol. Sci. 2024, 25(14), 7613; https://doi.org/10.3390/ijms25147613 Submission received: 30 April 2024 / Revised: 5 July 2024 / Accepted: 8 July 2024 / Published: 11 July 2024 (This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function) Downloadkeyboard_arrow_down Browse Figures Versions Notes Abstract The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His6-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His6-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked

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

Structural Dynamics of the <i>Bacillus subtilis</i> MntR Transcription Factor Is Locked by Mn²⁺ Binding

Manganese (II) ions are essential for a variety of bacterial cellular processes. The transcription factor MntR is a metallosensor that regulates Mn2+ ion homeostasis in the bacterium Bacillus subtilis. Its DNA-binding affinity is increased by Mn2+ ion binding, allowing it to act as a transcriptional repressor of manganese import systems. Although experimentally well-researched, the molecular mechanism that regulates this process is still a puzzle. Computational simulations supported by circular dichroism (CD), differential scanning calorimetry (DSC) and native gel electrophoresis (native-PAGE) experiments were employed to study MntR structural and dynamical properties in the presence and absence of Mn2+ ions. The results of molecular dynamics (MD) simulations revealed that Mn2+ ion binding reduces the structural dynamics of the MntR protein and shifts the dynamic equilibrium towards the conformations adequate for DNA binding. Results of CD and DSC measurements support the computational results showing the change in helical content and stability of the MntR protein upon Mn2+ ion binding. Further, MD simulations show that Mn2+ binding induces polarization of the protein electrostatic potential, increasing the positive electrostatic potential of the DNA-binding helices in particular. In order to provide a deeper understanding of the changes in protein structure and dynamics due to Mn2+ binding, a mutant in which Mn2+ binding is mimicked by a cysteine bridge was constructed and also studied computationally and experimentally

Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from <i>Helicobacter pylori</i>

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His6-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His6-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked

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