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1.
Int J Biol Macromol ; 226: 37-50, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36470440

RESUMO

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.


Assuntos
Helicobacter pylori , Domínio Catalítico , Sítios de Ligação , Helicobacter pylori/metabolismo , Adenilossuccinato Sintase/química , Adenilossuccinato Sintase/metabolismo , Inosina Monofosfato/química , Inosina Monofosfato/metabolismo , Conformação Proteica , Simulação de Dinâmica Molecular
2.
J Enzyme Inhib Med Chem ; 37(1): 1083-1097, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35437103

RESUMO

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.


Assuntos
Helicobacter pylori , Purina-Núcleosídeo Fosforilase , Sítios de Ligação , Técnicas de Cultura de Células , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Purinas/química , Purinas/farmacologia
3.
Sci Rep ; 11(1): 11144, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34045551

RESUMO

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.


Assuntos
Escherichia coli/genética , Mutação , Purina-Núcleosídeo Fosforilase/genética , Triptofano/genética , Sítios de Ligação , Escherichia coli/metabolismo , Modelos Moleculares , Conformação Proteica , Purina-Núcleosídeo Fosforilase/metabolismo
4.
Sci Rep ; 8(1): 15427, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30337572

RESUMO

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.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Nucleosídeos/metabolismo , Fosfatos/metabolismo , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Cinética , Ligantes , Modelos Moleculares , Mutação , Conformação Proteica , Purina-Núcleosídeo Fosforilase/genética , Especificidade por Substrato
5.
FEBS J ; 285(7): 1305-1325, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29430816

RESUMO

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (Pi ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes Pi binding, while a three-site model is needed to characterize FA binding, irrespective of Pi presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with Pi and FA shows, however, that Pi binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that Pi moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. DATABASES: The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). ENZYMES: Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463.


Assuntos
Helicobacter pylori/enzimologia , Conformação Proteica , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Estabilidade Enzimática , Formicinas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Simulação de Dinâmica Molecular , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Especificidade por Substrato , Temperatura
6.
J Am Soc Mass Spectrom ; 27(1): 73-82, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26337516

RESUMO

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.


Assuntos
Proteínas de Bactérias/química , Domínio Catalítico , Medição da Troca de Deutério/métodos , Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , Purina-Núcleosídeo Fosforilase/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Dados de Sequência Molecular , Fosfatos/química , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica , Purina-Núcleosídeo Fosforilase/metabolismo
7.
FEBS J ; 281(7): 1860-71, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24785777

RESUMO

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 phosphorylase - a homohexamer composed of three linked dimers - since 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 µm. 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. STRUCTURED DIGITAL ABSTRACT: ●PNP and PNP bind by molecular sieving (1, 2, 3, 4).


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , Multimerização Proteica , Purina-Núcleosídeo Fosforilase/química , Sequência de Aminoácidos , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Estabilidade Proteica , Purina-Núcleosídeo Fosforilase/genética
8.
Bioorg Med Chem ; 20(22): 6758-69, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23040896

RESUMO

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.


Assuntos
Purina-Núcleosídeo Fosforilase/metabolismo , Animais , Sítios de Ligação , Calorimetria , Domínio Catalítico , Bovinos , Fluorometria , Hipoxantina/química , Hipoxantina/metabolismo , Ligantes , Nucleosídeos de Purina/química , Nucleosídeos de Purina/metabolismo , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/genética , Pirimidinonas/química , Pirimidinonas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica
9.
FEBS Lett ; 586(7): 967-71, 2012 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-22569248

RESUMO

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.


Assuntos
Antineoplásicos/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Formicinas/metabolismo , Fosfatos/metabolismo , Purina-Núcleosídeo Fosforilase/química , Antineoplásicos/química , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Formicinas/química , Cinética , Ligantes , Modelos Moleculares , Concentração Osmolar , Fosfatos/química , Conformação Proteica , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Titulometria , Água/química , Água/metabolismo
10.
Biochimie ; 93(9): 1610-22, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21672603

RESUMO

The catalytic mechanism of Escherichia coli purine nucleoside phosphorylase (PNP) is revised using site-directed mutagenesis, kinetic studies and structure determinations. The experimental evidence on the role of the particular catalytic amino acid during catalysis has not been available. Therefore, the active site mutants Arg24Ala, Asp204Ala, Asp204Asn, Arg217Ala and Asp204Ala/Arg217Ala were prepared and their kinetics and thermodynamic studies were carried out. The activity tests with natural substrates and 7-methylguanosine confirmed the earlier hypothesis, that catalysis involves protonation of the purine base at position N7 by Asp204, which is triggered by Arg217. The crystal structures of the wild type in complexes with phosphate and sulphate, respectively, and of the Arg24Ala mutant in complex with phosphate/sulphate were determined. The structural data show that previously observed conformational change is a result of the phosphate binding and its interaction with Arg24. As E. coli PNP is a promising candidate for the tumour-directed gene therapy, our results may also help to design efficient mutants useful in gene therapy.


Assuntos
Escherichia coli/enzimologia , Purina-Núcleosídeo Fosforilase/química , Sítios de Ligação , Catálise , Cristalografia por Raios X , Escherichia coli/metabolismo , Guanosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
11.
Biochem Biophys Res Commun ; 391(1): 703-8, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19944078

RESUMO

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).


Assuntos
Inibidores Enzimáticos/química , Guanina/análogos & derivados , Organofosfonatos/química , Purina-Núcleosídeo Fosforilase/química , Proteínas Recombinantes/química , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/química , Glutamina/química , Glicina/química , Guanina/química , Guanina/farmacologia , Organofosfonatos/farmacologia , Fosfatos/química , Multimerização Proteica , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Proteínas Recombinantes/antagonistas & inibidores , Ribose/química
12.
J Phys Condens Matter ; (28): 285223, 2007 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-20126640

RESUMO

Green fluorescent protein (GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; i.e., the principles of microscopic reversibility do not apply. We have generated the mutant S65T/G67A-GFP, which is unable to form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of GdnHCl. The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of bis-ANS. Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements, that S65T/G67A-GFP folding is largely reversible. During equilibrium folding, the first step is formation of molten globule, prone to aggregation.

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