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Agnieszka Bzowska - One of the best experts on this subject based on the ideXlab platform.
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validation of the catalytic mechanism of escherichia coli purine nucleoside phosphorylase by structural and kinetic studies
Biochimie, 2011Co-Authors: Goran Mikleusevic, Agnieszka Bzowska, Zoran Stefanic, Marta Narczyk, Beata Wielguskutrowska, Marija LuicAbstract: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.
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interactions of calf spleen purine nucleoside phosphorylase with formycin b and its aglycone spectroscopic and kinetic studies
Nucleosides Nucleotides & Nucleic Acids, 2007Co-Authors: Jacek Wierzchowski, Beata Iwańska, Agnieszka Bzowska, David ShugarAbstract:Phosphorolysis of 7-Methylguanosine by calf spleen purine nucleoside phosphorylase (PNP) is weakly inhibited, uncompetitively, by Formycin B (FB) with K i = 100 μ M and more effectively by its aglycone (7KPP), IC50 35–100 μ M. In striking contrast, 7KPP inhibits the reverse reaction (synthesis of 8-azaguanosine from 8-azaguanine) competitively, with K i ∼ 2–4 μ M. Formycin B forms only a weakly fluorescent complex with PNP, and 7KPP even less so, indicating that both ligands bind as the neutral, not anionic, forms. 7KPP is a rare example of a PNP non-substrate inhibitor of both the phosphorolytic and reverse synthetic pathways.
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calf spleen purine nucleoside phosphorylase complex kinetic mechanism hydrolysis of 7 methylguanosine and oligomeric state in solution
Biochimica et Biophysica Acta, 2002Co-Authors: Agnieszka BzowskaAbstract:The active enzyme form was found to be a homotrimer, no active monomers were observed. Only in the presence of an extremely high orthophosphate concentration (0.5 M) or at a low enzyme concentration (0.2 μg/ml) with no ligands present a small fraction of the enzyme is probably in a dissociated and/or non-active form. The specific activity is invariant over a broad enzyme concentration range (0.017 μg/ml–0.29 mg/ml). At concentrations below 0.9 μg/ml and in the absence of ligands the enzyme tends to loose its catalytic activity, while in the presence of any substrate or at higher concentrations it was found to be active as a trimer. In the absence of phosphate the enzyme catalyses the hydrolysis of 7-Methylguanosine (m7Guo) with a catalytic rate constant 1.3×10−3 s−1 as compared with the rate of 38 s−1 for the phosphorolysis of this nucleoside. The initial pre-steady-state phase of the phosphorolysis of m7Guo, 70 s−1, is almost twice faster than the steady-state rate and indicates that the rate-limiting step is subsequent to the glycosidic bond cleavage. Complex kinetic behaviour with substrates of phosphorolytic direction (various nucleosides and orthophosphate) was observed; data for phosphate as the variable substrate with inosine and guanosine, but not with their 7-methyl counterparts, might be interpreted as two binding sites with different affinities, or as a negative cooperativity. However, the titration of the enzyme intrinsic fluorescence with 0.2 μM–30 mM phosphate is consistent with only one dissociation constant for phosphate, Kd=220±120 μM. Protective effects of ligands on the thermal inactivation of the enzyme indicate that all substrates of the phosphorolytic and the synthetic reactions are able to form binary complexes with the calf spleen purine nucleoside phosphorylase. The purine bases, guanine and hypoxanthine, bind strongly with dissociation constants of about 0.1 μM, while all other ligands studied, including 7-methylguanine and 7-methylhypoxanthine, bind at least 3 orders of magnitude less potently. Binding of guanine and hypoxanthine is about 10-fold weakened by the presence of phosphate. These observations are best interpretable by the complex kinetic mechanism of the phosphorolytic reaction involving (i) random substrate binding, (ii) unusually slow, hence strongly rate-limiting, dissociation of the products guanine and hypoxanthine, but not 7-methylguanine and 7-methylhypoxanthine, and (iii) dual function of the phosphate binding site with phosphate acting as a substrate and as a modifier helping in the release of a purine base after glycosidic bond cleavage.
Marzena Jankowskaanyszka - One of the best experts on this subject based on the ideXlab platform.
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synthesis of n2 modified 7 methylguanosine 5 monophosphates as nematode translation inhibitors
Bioorganic & Medicinal Chemistry, 2012Co-Authors: Karolina Piecyk, Richard E. Davis, Marzena JankowskaanyszkaAbstract:Abstract Preparative scale synthesis of 14 new N2-modified mononucleotide 5′ mRNA cap analogues was achieved. The key step involved use of an SNAr reaction with protected 2-fluoro inosine and various primary and secondary amines. The derivatives were tested in a parasitic nematode, Ascaris suum, cell-free system as translation inhibitors. The most effective compound with IC50 ∼0.9 μM was a N2-p-metoxybenzyl-7-Methylguanosine-5′-monophosphate 35.
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diverse role of three tyrosines in binding of the rna 5 cap to the human nuclear cap binding complex
Journal of Molecular Biology, 2009Co-Authors: Remigiusz Worch, Catherine Mazza, Stephen Cusack, Anna Niedzwiecka, Janusz Stepinski, Edward Darzynkiewicz, Marzena Jankowskaanyszka, Ryszard StolarskiAbstract:The heterodimeric nuclear cap-binding complex (CBC) specifically recognizes the monomethylguanosine 5′ cap structure of the eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is essential for nuclear maturation of mRNA, for nuclear export of U snRNA in metazoans, and for nonsense-mediated decay of mRNA and the pioneer round of translation. We analysed the recognition of the cap by native human CBC and mutants in which each tyrosine that stacks with the 7-Methylguanosine moiety was replaced by phenylalanine or alanine and both tyrosines were replaced by phenylalanines. The equilibrium association constants (Kas) for two selected cap analogues, P1-7-Methylguanosine-5′ P3-guanosine-5′ triphosphate and 7-Methylguanosine triphosphate, were determined by two independent methods, fluorescence titration and surface plasmon resonance. We could distinguish two tyrosines, Y43 and Y20, in stabilization of the cap inside the CBC-binding pocket. In particular, lack of Y20 in CBC leads to a greater affinity of the mono- than the dinucleotide cap analogue, in contrast to the wild-type protein. A crucial role of cation–π stacking in the mechanism of the specific cap recognition by CBC was postulated from the comparison of the experimentally derived Gibbs free binding energy (ΔG°) with the stacking energy (ΔE) of the 7-Methylguanosine/Y binary and ternary complexes calculated by the Moller–Plesset second-order perturbation method. The resulting kinetic model of the association between the capped RNA and CBC, based on the experimental data and quantum calculations, is discussed with respect to the “CBC-to-eukaryotic initiation factor 4E handoff” of mRNA.
Wenbin Zhou - One of the best experts on this subject based on the ideXlab platform.
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arabidopsis trm5 encodes a nuclear localised bifunctional trna guanine and inosine n1 methyltransferase that is important for growth
PLOS ONE, 2019Co-Authors: Pei Qin Ng, H. Wang, R. David, P. Mittal, R. Bock, Jun Li, Jing Zhao, Trung Do, Ming Zhao, Wenbin ZhouAbstract:Modified nucleosides in tRNAs are critical for protein translation. N1-methylguanosine-37 and N1-methylinosine-37 in tRNAs, both located at the 3’-adjacent to the anticodon, are formed by Trm5. Here we describe Arabidopsis thaliana AtTRM5 (At3g56120) as a Trm5 ortholog. Attrm5 mutant plants have overall slower growth as observed by slower leaf initiation rate, delayed flowering and reduced primary root length. In Attrm5 mutants, mRNAs of flowering time genes are less abundant and correlated with delayed flowering. We show that AtTRM5 complements the yeast trm5 mutant, and in vitro methylates tRNA guanosine-37 to produce N1-methylguanosine (m1G). We also show in vitro that AtTRM5 methylates tRNA inosine-37 to produce N1-methylinosine (m1I) and in Attrm5 mutant plants, we show a reduction of both N1-methylguanosine and N1-methylinosine. We also show that AtTRM5 is localized to the nucleus in plant cells. Proteomics data showed that photosynthetic protein abundance is affected in Attrm5 mutant plants. Finally, we show tRNA-Ala aminoacylation is not affected in Attrm5 mutants. However the abundance of tRNA-Ala and tRNA-Asp 5’ half cleavage products are deduced. Our findings highlight the bifunctionality of AtTRM5 and the importance of the post-transcriptional tRNA modifications m1G and m1I at tRNA position 37 in general plant growth and development.
Ryszard Stolarski - One of the best experts on this subject based on the ideXlab platform.
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diverse role of three tyrosines in binding of the rna 5 cap to the human nuclear cap binding complex
Journal of Molecular Biology, 2009Co-Authors: Remigiusz Worch, Catherine Mazza, Stephen Cusack, Anna Niedzwiecka, Janusz Stepinski, Edward Darzynkiewicz, Marzena Jankowskaanyszka, Ryszard StolarskiAbstract:The heterodimeric nuclear cap-binding complex (CBC) specifically recognizes the monomethylguanosine 5′ cap structure of the eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is essential for nuclear maturation of mRNA, for nuclear export of U snRNA in metazoans, and for nonsense-mediated decay of mRNA and the pioneer round of translation. We analysed the recognition of the cap by native human CBC and mutants in which each tyrosine that stacks with the 7-Methylguanosine moiety was replaced by phenylalanine or alanine and both tyrosines were replaced by phenylalanines. The equilibrium association constants (Kas) for two selected cap analogues, P1-7-Methylguanosine-5′ P3-guanosine-5′ triphosphate and 7-Methylguanosine triphosphate, were determined by two independent methods, fluorescence titration and surface plasmon resonance. We could distinguish two tyrosines, Y43 and Y20, in stabilization of the cap inside the CBC-binding pocket. In particular, lack of Y20 in CBC leads to a greater affinity of the mono- than the dinucleotide cap analogue, in contrast to the wild-type protein. A crucial role of cation–π stacking in the mechanism of the specific cap recognition by CBC was postulated from the comparison of the experimentally derived Gibbs free binding energy (ΔG°) with the stacking energy (ΔE) of the 7-Methylguanosine/Y binary and ternary complexes calculated by the Moller–Plesset second-order perturbation method. The resulting kinetic model of the association between the capped RNA and CBC, based on the experimental data and quantum calculations, is discussed with respect to the “CBC-to-eukaryotic initiation factor 4E handoff” of mRNA.
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the antiviral drug ribavirin does not mimic the 7 methylguanosine moiety of the mrna cap structure in vitro
RNA, 2005Co-Authors: Belinda J Westman, Remigiusz Worch, Janusz Stepinski, Edward Darzynkiewicz, Ryszard Stolarski, Lisa Beeren, Ewa Grudzien, Joanna Zuberek, Jacek Jemielity, Robert E. RhoadsAbstract:The eukaryotic initiation factor eIF4E binds the mRNA 5' cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study (Kentsis et al. 2004) presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4E-mediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m(7)GTP and m(7)GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap.
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Significance of the first transcribed nucleoside of capped RNA for ligand-induced folding of the cap-binding complex
Journal of Physics: Condensed Matter, 2005Co-Authors: Remigiusz Worch, Catherine Mazza, Stephen Cusack, Anna Niedzwiecka, Janusz Stepinski, Marzena Jankowska-anyszka, Edward Darzynkiewicz, Ryszard StolarskiAbstract:Many proteins, including those that bind RNA, change conformation upon binding a ligand, a phenomenon known as induced fit. CBP20, the small subunit of the nuclear cap-binding complex (CBC), recognizes specifically the 5' cap of eukaryotic mRNA and snRNA. The N- and C-terminal regions of the CBP20 subunit of the human nuclear cap-binding complex only acquire a proper fold in complex with capped RNA. The cap is composed of 7-Methylguanosine linked by a 5'-to-5' triphosphate bridge to the first transcribed nucleoside of the RNA. The significance of the latter for the capped RNA-CBC association and local folding of CBC has been characterized by emission spectroscopy. Fluorescence titration of CBC has been performed for three selected, mono- and dinucleotide mRNA 5' cap analogues. The measured values of the equilibrium association constant and the corresponding Gibbs free energy depend on the type of the first transcribed nucleoside (purine or pyrimidine), and decrease ∼10-fold in the case of a mononucleotide analogue, 7-Methylguanosine triphosphate. However, the total quenching of the intrinsic protein fluorescence is similar for each analogue. Changes of the solvent-accessible CBC hydrophobic surface of CBC on binding of the structurally different cap analogues have been followed using bis-ANS fluorescent probe.
Nathaniel J. Moorman - One of the best experts on this subject based on the ideXlab platform.
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Human cytomegalovirus TRS1 protein associates with the 7-Methylguanosine mRNA cap and facilitates translation.
Proteomics, 2015Co-Authors: Benjamin Ziehr, Erik M. Lenarcic, Heather A. Vincent, Chad Cecil, Benjamin A. Garcia, Thomas Shenk, Nathaniel J. MoormanAbstract:Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host/pathogen interface. In this study, we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-Methylguanosine mRNA cap, increased the total level of protein synthesis, and colocalized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a nonviral reporter gene and increased the translation of a reporter containing an HCMV 5' untranslated region (5'UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5'UTR required the pTRS1 RNA and double-stranded RNA-dependent protein kinase (PKR)-binding domains, and was likely the result of PKR inhibition. However, pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5'UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms.