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  • 151.
    Pang, Yanhong
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kovachev, Petar Stefanov
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Macedo, Bruno
    Faculdade de Farmácia, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Bloco B, Subsolo, Sala 17, Rio de Janeiro, Brazil.
    Westermark, Gunilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Intervention of ribosomal RNA in HET-s prion aggregation Intervention of ribosomal RNA in HET-s prion aggregationManuscript (preprint) (Other academic)
    Abstract [en]

    The role of nucleic acids in prion aggregation / disaggregation has remained unclear. Here, using HET-s prion from Podospora anserina as a model system, we have studied the role of RNA, particularly different domains of ribosomal RNA, in its aggregation process. Our results show that domain V rRNA, from the large subunit of the ribosome, substantially prevents amyloid aggregation of the HET-s prion in a concentration dependent manner. Instead, it promotes the formation of oligomeric seeds, which facilitate de novo HET-s aggregation. The interaction sites for the HET-s prion on domain V rRNA were also identified and shown to overlap with the sites previously found to responsible for the protein folding activity of the ribosome (PFAR). This study provides a missing link between the role of rRNA-based PFAR and prion propagation.

  • 152.
    Pang, Yanhong
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kurella, Sriram
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Voisset, Cecile
    Samanta, Dibyendu
    Banerjee, Debapriya
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Schabe, Ariane
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Das Gupta, Chanchal
    Galons, Herve
    Blondel, Marc
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 26, p. 19081-19089Article in journal (Refereed)
    Abstract [en]

    Domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active center for the protein folding activity of the ribosome (PFAR). Using in vitro transcribed domain V rRNAs from Escherichia coli and Saccharomyces cerevisiae as the folding modulators and human carbonic anhydrase as a model protein, we demonstrate that PFAR is conserved from prokaryotes to eukaryotes. It was shown previously that 6-aminophenanthridine (6AP), an antiprion compound, inhibits PFAR. Here, using UV cross-linking followed by primer extension, we show that the protein substrates and 6AP interact with a common set of nucleotides on domain V of 23S rRNA. Mutations at the interaction sites decreased PFAR and resulted in loss or change of the binding pattern for both the protein substrates and 6AP. Moreover, kinetic analysis of human carbonic anhydrase refolding showed that 6AP decreased the yield of the refolded protein but did not affect the rate of refolding. Thus, we conclude that 6AP competitively occludes the protein substrates from binding to rRNA and thereby inhibits PFAR. Finally, we propose a scheme clarifying the mechanism by which 6AP inhibits PFAR.

  • 153.
    Pati, Sarah G.
    et al.
    Eawag, Swiss Fed Inst Aquat Sci & Technol, CH-8600 Dubendorf, Switzerland.;Swiss Fed Inst Technol, Inst Biogeochem & Pollutant Dynam IBP, CH-8092 Zurich, Switzerland..
    Kohler, Hans-Peter E.
    Eawag, Swiss Fed Inst Aquat Sci & Technol, CH-8600 Dubendorf, Switzerland..
    Pabis, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Lodz Univ Technol, Inst Appl Radiat Chem, PL-90924 Lodz, Poland..
    Paneth, Piotr
    Lodz Univ Technol, Inst Appl Radiat Chem, PL-90924 Lodz, Poland..
    Parales, Rebecca E.
    Univ Calif Davis, Dept Microbiol & Mol Genet, Davis, CA 95616 USA..
    Hofstetter, Thomas B.
    Eawag, Swiss Fed Inst Aquat Sci & Technol, CH-8600 Dubendorf, Switzerland.;Swiss Fed Inst Technol, Inst Biogeochem & Pollutant Dynam IBP, CH-8092 Zurich, Switzerland..
    Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants2016In: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 50, no 13, p. 6708-6716Article in journal (Refereed)
    Abstract [en]

    Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent C-13- and H-2-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O-2 activation and aromatic C oxygenation. The contribution of enzymatic O-2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O-2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants.

  • 154.
    Pavlov, Michael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Liljas, Anders
    Dept Biochem & Struct Biol, Lund University..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    A recent intermezzo at the Ribosome Club2017In: Philosophical Transactions of the Royal Society of London. Biological Sciences, ISSN 0962-8436, E-ISSN 1471-2970, Vol. 372, no 1716, article id 20160185Article, review/survey (Refereed)
    Abstract [en]

    Two sets of ribosome structures have recently led to two different interpretations of what limits the accuracy of codon translation by transfer RNAs. In this review, inspired by this intermezzo at the Ribosome Club, we briefly discuss accuracy amplification by energy driven proofreading and its implementation in genetic code translation. We further discuss general ways by which the monitoring bases of 16S rRNA may enhance the ultimate accuracy (d-values) and how the codon translation accuracy is reduced by the actions of Mg2+ ions and the presence of error inducing aminoglycoside antibiotics. We demonstrate that complete freezing-in of cognate-like tautomeric states of ribosome-bound nucleotide bases in transfer RNA or messenger RNA is not compatible with recent experiments on initial codon selection by transfer RNA in ternary complex with elongation factor Tu and GTP. From these considerations, we suggest that the sets of 30S subunit structures from the Ramakrishnan group and 70S structures from the Yusupov/Yusupova group may, after all, reflect two sides of the same coin and how the structurally based intermezzo at the Ribosome Club may be resolved simply by taking the dynamic aspects of ribosome function into account. This article is part of the themed issue 'Perspectives on the ribosome'.

  • 155.
    Pavlov, Michael Y.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Optimal control of gene expression for fast proteome adaptation to environmental change2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 51, p. 20527-20532Article in journal (Refereed)
    Abstract [en]

    Bacterial populations growing in a changing world must adjust their proteome composition in response to alterations in the environment. Rapid proteome responses to growth medium changes are expected to increase the average growth rate and fitness value of these populations. Little is known about the dynamics of proteome change, e. g., whether bacteria use optimal strategies of gene expression for rapid proteome adjustments and if there are lower bounds to the time of proteome adaptation in response to growth medium changes. To begin answering these types of questions, we modeled growing bacteria as stoichiometrically coupled networks of metabolic pathways. These are balanced during steady-state growth in a constant environment but are initially unbalanced after rapid medium shifts due to a shortage of enzymes required at higher concentrations in the new environment. We identified an optimal strategy for rapid proteome adjustment in the absence of protein degradation and found a lower bound to the time of proteome adaptation after medium shifts. This minimal time is determined by the ratio between the Kullback-Leibler distance from the pre- to the postshift proteome and the postshift steady-state growth rate. The dynamics of optimally controlled proteome adaptation has a simple analytical solution. We used detailed numerical modeling to demonstrate that realistic bacterial control systems can emulate this optimal strategy for rapid proteome adaptation. Our results may provide a conceptual link between the physiology and population genetics of growing bacteria.

  • 156.
    Pavlov, Michael Y.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Initiation of bacterial protein synthesis with wild type and novel mutants of initiation factor2011In: Ribosomes: Structure, Function and Dynamics / [ed] Marina Rodnina, Wolfgang Wintermeyer, Rachel Green, Springer-Verlag New York, 2011, p. 129-141Conference paper (Refereed)
  • 157.
    Pavlov, Michael Y.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Zorzet, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits2011In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 30, no 2, p. 289-301Article in journal (Refereed)
    Abstract [en]

    We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNA(i)). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNA(i), implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNA(i) and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10(-4) for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases.

     

  • 158.
    Petrovic, Dusan
    et al.
    Forschungszentrum Julich, Inst Complex Syst Struct Biochem, D-52425 Julich, Germany..
    Frank, David
    Rhein Westfal TH Aachen, Inst Mol Biotechnol, Worringerweg 1, D-52074 Aachen, Germany.;Aquila Biolabs GmbH, Arnold Sommerfeld Ring 2, D-52499 Baesweiler, Germany..
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Hoffmann, Kurt
    Rhein Westfal TH Aachen, Inst Mol Biotechnol, Worringerweg 1, D-52074 Aachen, Germany..
    Strodel, Birgit
    Forschungszentrum Julich, Inst Complex Syst Struct Biochem, D-52425 Julich, Germany.;Heinrich Heine Univ Dusseldorf, Inst Theoret & Computat Chem, Univ Str 1, D-40225 Dusseldorf, Germany..
    Shuffling Active Site Substate Populations Affects Catalytic Activity: The Case of Glucose Oxidase2017In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 7, no 9, p. 6188-6197Article in journal (Refereed)
    Abstract [en]

    Glucose oxidase has wide applications in the pharmaceutical, chemical, and food industries. Many recent studies have enhanced key properties of this enzyme using directed evolution, yet without being able to reveal why these mutations are actually beneficial. This work presents a synergistic combination of experimental and computational methods, indicating how mutations, even when distant from the active site, positively affect glucose oxidase catalysis. We have determined the crystal structures of glucose oxidase mutants containing molecular oxygen in the active site. The catalytically important His516 residue has been previously shown to be flexible in the wild-type enzyme. The molecular dynamics simulations performed in this work allow us to quantify this floppiness, revealing that His516 exists in two states: catalytic and noncatalytic. The relative populations of these two substates are almost identical in the wild-type enzyme, with His516 readily shuffling between them. In the glucose oxidase mutants, on the other hand, the mutations enrich the catalytic His516 conformation and reduce the flexibility of this residue, leading to an enhancement in their catalytic efficiency. This study stresses the benefit of active site preorganization with respect to enzyme conversion rates by reducing molecular reorientation needs. We further suggest that the computational approach based on Hamiltonian replica exchange molecular dynamics, used in this study, may be a general approach to screening in silico for improved enzyme variants involving flexible catalytic residues.

  • 159.
    Petrovic, Dusan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Szeler, Klaudia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Challenges and advances in the computational modeling of biological phosphate hydrolysis2018In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 54, no 25, p. 3077-3089Article, review/survey (Refereed)
    Abstract [en]

    Phosphate ester hydrolysis is fundamental to many life processes, and has been the topic of substantial experimental and computational research effort. However, even the simplest of phosphate esters can be hydrolyzed through multiple possible pathways that can be difficult to distinguish between, either experimentally, or computationally. Therefore, the mechanisms of both the enzymatic and non-enzymatic reactions have been historically controversial. In the present contribution, we highlight a number of technical issues involved in reliably modeling these computationally challenging reactions, as well as proposing potential solutions. We also showcase examples of our own work in this area, discussing both the non-enzymatic reaction in aqueous solution, as well insights obtained from the computational modeling of organophosphate hydrolysis and catalytic promiscuity amongst enzymes that catalyze phosphoryl transfer. 

  • 160.
    Poberznik, Matic
    et al.
    Jozef Stefan Inst, Dept Phys & Organ Chem, Jamova Cesta 39, SI-1000 Ljubljana, Slovenia..
    Purg, Miha
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Repic, Matej
    Natl Inst Chem, Lab Biocomp & Bioinformat, Hajdrihova Ulica 19, SI-1000 Ljubljana, Slovenia..
    Mavri, Janez
    Natl Inst Chem, Lab Biocomp & Bioinformat, Hajdrihova Ulica 19, SI-1000 Ljubljana, Slovenia..
    Vianello, Robert
    Rudjer Boskovic Inst, Computat Organ Chem & Biochem Grp, Bijenicka Cesta 54, HR-10000 Zagreb, Croatia..
    Empirical Valence Bond Simulations of the Hydride-Transfer Step in the Monoamine Oxidase A Catalyzed Metabolism of Noradrenaline2016In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 120, no 44, p. 11419-11427Article in journal (Refereed)
    Abstract [en]

    Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines, such as dopamine, serotonin, and noradrenaline (NA), which is why they have been extensively implicated in the etiology and course of various neurodegenerative disorders and, accordingly, used as primary pharmacological targets to treat these debilitating cognitive diseases. The precise chemical mechanism through which MAOs regulate the amine Concentration, which is vital for the development of novel inhibitors, is still not unambiguously determined in the literature. In this work, we present atomistic empirical valence bond simulations of the rate-limiting step of the MAO-A-catalyzed NA (norepinephrine) degradation, involving hydride transfer from the substrate alpha-methylene group to the flavin moiety of the flavin adenine dinucleotide prosthetic group, employing the full dimensionality and thermal fluctuations of the hydrated enzyme, with extensive configurational sampling. We show that MAO-A lowers the free energy of activation by 14.3 kcal mol(-1) relative to that of the same reaction in aqueous solution, whereas the calculated activation free energy of Delta G(double dagger) = 20.3 +/- 1.6 kcal mori is found to be in reasonable agreement with the correlated experimental value of 16.5 kcal mol(-1). The results presented here strongly support the fact that both MAO-A and MAO-B isoforms function by the same hydride-transfer mechanism. We also considered a few point mutations of the "aromatic cage" tyrosine residue (Tyr444Phe, Tyr444Leu, Tyr444Trp, Tyr444His, and Tyr444Glu), and the calculated changes in the reaction barriers are in agreement with the experimental values, thus providing further support to the proposed mechanism.

  • 161.
    Punekar, Avinash S.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ribosomal RNA Modification Enzymes: Structural and functional studies of two methyltransferases for 23S rRNA modification in Escherichia coli 2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Escherichia coli ribosomal RNA (rRNA) is post-transcriptionally modified by site-specific enzymes. The role of most modifications is not known and little is known about how these enzymes recognize their target substrates. In this thesis, we have structurally and functionally characterized two S-adenosyl-methionine (SAM) dependent 23S rRNA methyltransferases (MTases) that act during the early stages of ribosome assembly in E. coli.

    RlmM methylates the 2'O-ribose of C2498 in 23S rRNA. We have solved crystal structures of apo RlmM at 1.9Å resolution and of an RlmM-SAM complex at 2.6Å resolution. The RlmM structure revealed an N-terminal THUMP domain and a C-terminal catalytic Rossmann-fold MTase domain. A continuous patch of conserved positive charge on the RlmM surface is likely used for RNA substrate recognition. The SAM-binding site is open and shallow, suggesting that the RNA substrate may be required for tight cofactor binding. Further, we have shown RlmM MTase activity on in vitro transcribed 23S rRNA and its domain V.

    RlmJ methylates the exocyclic N6 atom of A2030 in 23S rRNA. The 1.85Å crystal structure of RlmJ revealed a Rossmann-fold MTase domain with an inserted small subdomain unique to the RlmJ family. The 1.95Å structure of the RlmJ-SAH-AMP complex revealed that ligand binding induces structural rearrangements in the four loop regions surrounding the active site. The active site of RlmJ is similar to N6-adenine DNA MTases. We have shown RlmJ MTase activity on in vitro transcribed 23S rRNA and a minimal substrate corresponding to helix 72, specific for adenosine. Mutagenesis experiments show that residues Y4, H6, K18 and D164 are critical for catalytic activity.

    These findings have furthered our understanding of the structure, evolution, substrate recognition and mechanism of rRNA MTases.

    List of papers
    1. Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA
    Open this publication in new window or tab >>Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA
    Show others...
    2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 20, p. 10507-20Article in journal (Refereed) Published
    Abstract [en]

    RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2'O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM-AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2'O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate.

    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-187880 (URN)10.1093/nar/gks727 (DOI)000310970700054 ()22923526 (PubMedID)
    Available from: 2012-12-11 Created: 2012-12-11 Last updated: 2019-01-25Bibliographically approved
    2. Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli
    Open this publication in new window or tab >>Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli
    2013 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 69, p. 1001-1003Article in journal (Refereed) Published
    Abstract [en]

    Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni2+-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 angstrom resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 angstrom, beta = 104 degrees. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 angstrom(3) Da(-1).

    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-208048 (URN)10.1107/S1744309113020289 (DOI)000323719700010 ()
    Available from: 2013-09-24 Created: 2013-09-23 Last updated: 2017-12-06Bibliographically approved
    3. Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ
    Open this publication in new window or tab >>Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ
    Show others...
    2013 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 20, p. 9537-9548Article in journal (Refereed) Published
    Abstract [en]

    RlmJ catalyzes the m(6)A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m(6)A MTases than to RNA m(6)2A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.

    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-211566 (URN)10.1093/nar/gkt719 (DOI)000326746400036 ()23945937 (PubMedID)
    Available from: 2013-11-26 Created: 2013-11-26 Last updated: 2019-01-25Bibliographically approved
  • 162.
    Punekar, Avinash S.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli2013In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 69, p. 1001-1003Article in journal (Refereed)
    Abstract [en]

    Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni2+-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 angstrom resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 angstrom, beta = 104 degrees. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 angstrom(3) Da(-1).

  • 163.
    Punekar, Avinash S
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Shepherd, Tyson R
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Liljeruhm, Josefine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony C
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 20, p. 10507-20Article in journal (Refereed)
    Abstract [en]

    RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2'O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM-AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2'O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate.

  • 164.
    Purg, Miha
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elias, Mikael
    Univ Minnesota, Dept Biochem Mol Biol & Biophys, 1479 Gortner Ave, St Paul, MN 55108 USA.;Univ Minnesota, Biotechnol Inst, 1479 Gortner Ave, St Paul, MN 55108 USA..
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Similar Active Sites and Mechanisms Do Not Lead to Cross-Promiscuity in Organophosphate Hydrolysis: Implications for Biotherapeutic Engineering2017In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 139, no 48, p. 17533-17546Article in journal (Refereed)
    Abstract [en]

    Organophosphate hydrolases are proficient catalysts of the breakdown of neurotoxic organophosphates and have great potential as both biotherapeutics for treating acute organophosphate toxicity and as bioremediation agents. However, proficient organophosphatases such as serum paraoxonase 1 (PON1) and the organophosphate-hydrolyzing lactonase SsoPox are unable to hydrolyze bulkyorganophosphates with challenging leaving groups such as diisopropyl fluorophosphate (DFP) or venomous agent X, creating a major challenge for enzyme design. Curiously, despite their mutually exclusive substrate specificities, PON1 and diisopropyl fluorophosphatase (DFPase) have essentially identical active sites and tertiary structures. In the present work, we use empirical valence bond simulations to probe the catalytic mechanism of DFPase as well as temperature, pH, and mutational effects, demonstrating that DFPase and PON1 also likely utilize identical catalytic mechanisms to hydrolyze their respective substrates. However, detailed examination of both static structures and dynamical simulations demonstrates subtle but significant differences in the electrostatic properties and solvent penetration of the two active sites and, most critically, the role of residues that make no direct contact with either substrate in acting as "specificity switches" between the two enzymes. Specifically, we demonstrate that key residues that are structurally and functionally critical for the paraoxonase activity of PON1 prevent it from being able to hydrolyze DFP with its fluoride leaving group. These insights expand our understanding of the drivers of the evolution of divergent substrate specificity in enzymes with identical active sites and guide the future design of organophosphate hydrolases that hydrolyze compounds with challenging leaving groups.

  • 165.
    Purg, Miha
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pabis, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Baier, Florian
    Tokuriki, Nobuhiki
    Jackson, Colin
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Probing the mechanisms for the selectivity and promiscuity of methyl parathion hydrolase.2016In: Philosophical Transactions of the Royal Society of London A, Mathematical, Physical and Engineering Sciences, Vol. 374, p. 20160150-Article in journal (Refereed)
    Abstract [en]

    Diverse organophosphate hydrolases have convergently evolved the ability to hydrolyse man-made organophosphates. Thus, these enzymes are attractive model systems for studying the factors shaping enzyme functional evolution. Methyl parathion hydrolase (MPH) is an enzyme from the metallo-β-lactamase superfamily, which hydrolyses a wide range of organophosphate, aryl ester and lactone substrates. In addition, MPH demonstrates metal-ion-dependent selectivity patterns. The origins of this remain unclear, but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. Here, we present detailed mechanistic studies of the paraoxonase and arylesterase activities of MPH complexed with five different transition metal ions, and demonstrate that the hydrolysis reactions proceed via similar pathways and transition states. However, while it is possible to discern a clear structural origin for the selectivity between different substrates, the selectivity between different metal ions appears to lie instead in the distinct electrostatic properties of the metal ions themselves, which causes subtle changes in transition state geometries and metal–metal distances at the transition state rather than significant structural changes in the active site. While subtle, these differences can be significant for shaping the metal-ion-dependent activity patterns observed for this enzyme.

    This article is part of the themed issue ‘Multiscale modelling at the physics–chemistry–biology interface’.

  • 166. Quax, Tessa E. F.
    et al.
    Wolf, Yuri I.
    Koehorst, Jasper J.
    Wurtzel, Omri
    van der Oost, Richard
    Ran, Wenqi
    Blombach, Fabian
    Makarova, Kira S.
    Brouns, Stan J. J.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wagner, Gerhart
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sorek, Rotem
    Koonin, Eugene V.
    van der Oost, John
    Differential Translation Tunes Uneven Production of Operon-Encoded Proteins2013In: Cell Reports, ISSN 2211-1247, Vol. 4, no 5, p. 938-944Article in journal (Refereed)
    Abstract [en]

    Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins.

  • 167. Read, Randy J.
    et al.
    Adams, Paul D.
    Arendall, W. Bryan, III
    Brunger, Axel T.
    Emsley, Paul
    Joosten, Robbie P.
    Kleywegt, Gerard J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Krissinel, Eugene B.
    Luetteke, Thomas
    Otwinowski, Zbyszek
    Perrakis, Anastassis
    Richardson, Jane S.
    Sheffler, William H.
    Smith, Janet L.
    Tickle, Ian J.
    Vriend, Gert
    Zwart, Peter H.
    A New Generation of Crystallographic Validation Tools for the Protein Data Bank2011In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 19, no 10, p. 1395-1412Article in journal (Refereed)
    Abstract [en]

    This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a now assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators.

  • 168. Reddy, B. K. Kishore
    et al.
    Landge, Sudhir
    Ravishankar, Sudha
    Patil, Vikas
    Shinde, Vikas
    Tantry, Subramanyam
    Kale, Manoj
    Raichurkar, Anandkumar
    Menasinakai, Sreenivasaiah
    Mudugal, Naina Vinay
    Ambady, Anisha
    Ghosh, Anirban
    Tunduguru, Ragadeepthi
    Kaur, Parvinder
    Singh, Ragini
    Kumar, Naveen
    Bharath, Sowmya
    Sundaram, Aishwarya
    Bhat, Jyothi
    Sambandamurthy, Vasan K.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Das, Kaveri
    Bandodkar, Balachandra
    Malolanarasimhan, Krishnan
    Mukherjee, Kakoli
    Ramachandran, Vasanthi
    Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors2014In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 58, no 6, p. 3312-3326Article in journal (Refereed)
    Abstract [en]

    Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis.

  • 169.
    Reuveni, Shlomi
    et al.
    Harvard Univ, Dept Syst Biol, HMS, 200 Longwood Ave, Boston, MA 02115 USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Paulsson, Johan
    Harvard Univ, Dept Syst Biol, HMS, 200 Longwood Ave, Boston, MA 02115 USA..
    Ribosomes are optimized for autocatalytic production2017In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 547, no 7663, p. 293-297Article in journal (Refereed)
    Abstract [en]

    Many fine-scale features of ribosomes have been explained in terms of function, revealing a molecular machine that is optimized for error-correction, speed and control. Here we demonstrate mathematically that many less well understood, larger-scale features of ribosomes-such as why a few ribosomal RNA molecules dominate the mass and why the ribosomal protein content is divided into 55-80 small, similarly sized segments-speed up their autocatalytic production.

  • 170.
    Risso, Valeria A.
    et al.
    Univ Granada, Fac Ciencias, Dept Quim Fis, E-18071 Granada, Spain..
    Martinez-Rodriguez, Sergio
    Univ Granada, Fac Ciencias, Dept Quim Fis, E-18071 Granada, Spain..
    Candel, Adela M.
    Univ Granada, Fac Ciencias, Dept Quim Fis, E-18071 Granada, Spain..
    Krüger, Dennis M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pantoja-Uceda, David
    CSIC, Inst Quim Fis Rocasolano, Dept Quim Fis Biol, C Serrano 119, E-28006 Madrid, Spain..
    Ortega-Munoz, Mariano
    Univ Granada, Fac Ciencias, Dept Quim Organ, E-18071 Granada, Spain..
    Santoyo-Gonzalez, Francisco
    Univ Granada, Fac Ciencias, Dept Quim Organ, E-18071 Granada, Spain..
    Gaucher, Eric A.
    Georgia Inst Technol, Sch Biol, Sch Chem & Biochem, Parker H Petit Inst Bioengn & Biosci, Atlanta, GA 30322 USA..
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bruix, Marta
    CSIC, Inst Quim Fis Rocasolano, Dept Quim Fis Biol, C Serrano 119, E-28006 Madrid, Spain..
    Gavira, Jose A.
    Univ Granada, CSIC, Inst Andaluz Ciencias Tierra, Lab Estudios Cristalog, Ave la Palmeras 4, Granada 18100, Armilla, Spain..
    Sanchez-Ruiz, Jose M.
    Univ Granada, Fac Ciencias, Dept Quim Fis, E-18071 Granada, Spain..
    De novo active sites for resurrected Precambrian enzymes2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 16113Article in journal (Refereed)
    Abstract [en]

    Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

  • 171.
    Russo, Francesco
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Åkerbladh, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Roos, Annette K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Naworyta, Agata
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sokolowski, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Henderson, Ian
    Alling, Torey
    Bailey, Mai A.
    Files, Megan
    Parish, Tanya
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents2015In: ChemistryOpen, ISSN 2191-1363, Vol. 4, no 3, p. 342-362Article in journal (Refereed)
    Abstract [en]

    This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure-activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs.

  • 172. Sacco, Emmanuelle
    et al.
    Slama, Nawel
    Bäckbro, Kristina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Parish, Tanya
    Laval, Francoise
    Daffe, Mamadou
    Eynard, Nathalie
    Quemard, Annaik
    Revisiting the Assignment of Rv0241c to Fatty Acid Synthase Type II of Mycobacterium tuberculosis2010In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 192, no 15, p. 4037-4044Article in journal (Refereed)
    Abstract [en]

    The fatty acid synthase type II enzymatic complex of Mycobacterium tuberculosis (FAS-IIMt) catalyzes an essential metabolic pathway involved in the biosynthesis of major envelope lipids, mycolic acids. The partner proteins of this singular FAS-II system represent relevant targets for antituberculous drug design. Two heterodimers of the hydratase 2 protein family, HadAB and HadBC, were shown to be involved in the (3R)-hydroxyacyl-ACP dehydration (HAD) step of FAS-IIMt cycles. Recently, an additional member of this family, Rv0241c, was proposed to have the same function, based on the heterologous complementation of a HAD mutant of the yeast mitochondrial FAS-II system. In the present work, Rv0241c was able to complement a HAD mutant in the Escherichia coli model but not a dehydratase-isomerase deficient mutant. However, an enzymatic study of the purified protein demonstrated that Rv0241c possesses a broad chain length specificity for the substrate, unlike FAS-IIMt enzymes. Most importantly, Rv0241c exhibited a strict dependence on the coenzyme A (CoA) as opposed to AcpM, the natural acyl carrier protein bearing the chains elongated by FAS-IIMt. The deletion of Rv0241c showed that this gene is not essential to M. tuberculosis survival in vitro. The resulting mutant did not display any change in the mycolic acid profile. This demonstrates that Rv0241c is a trans-2-enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase that does not belong to FAS-IIMt. The relevance of a heterologous complementation strategy to identifying proteins of such a system is questioned.

  • 173.
    Selmer, Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gao, Yong-Gui
    Weixlbaumer, Albert
    Ramakrishnan, V.
    Ribosome engineering to promote new crystal forms2012In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 68, no 5, p. 578-583Article in journal (Refereed)
    Abstract [en]

    Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in similar to the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.

  • 174.
    Shepherd, Tyson R
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. MIT, Dept Biol Engn, Cambridge, MA 02139 USA..
    Du, Liping
    Vanderbilt Univ, Med Ctr, Dept Pharmacol, Nashville, TN 37232 USA.;Vanderbilt Univ, Med Ctr, Ctr Quantitat Sci, Nashville, TN 37232 USA..
    Liljeruhm, Josefine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Samudyata,
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wang, Jinfan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    Sjödin, Marcus O.D.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Swedish Toxicol Sci Res Ctr Swetox, S-15136 Sodertalje, Sweden..
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. GE Healthcare Biosci, Life Sci, S-75184 Uppsala, Sweden..
    Yomo, Tetsuya
    East China Normal Univ, Inst Biol & Informat Sci, Sch Comp Sci & Software Engn, Sch Life Sci, Shanghai 200062, Peoples R China..
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Vanderbilt Univ, Med Ctr, Dept Pharmacol, Nashville, TN 37232 USA..
    De novo design and synthesis of a 30-cistron translation-factor module2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 18, p. 10895-10905Article in journal (Refereed)
    Abstract [en]

    Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.

  • 175. Shurki, Avital
    et al.
    Derat, Etienne
    Barrozo, Alexandre
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    How valence bond theory can help you understand your (bio)chemical reaction2015In: Chemical Society Reviews, ISSN 0306-0012, E-ISSN 1460-4744, Vol. 44, no 5, p. 1037-1052Article, review/survey (Refereed)
    Abstract [en]

    Almost a century has passed since valence bond (VB) theory was originally introduced to explain covalent bonding in the H-2 molecule within a quantum mechanical framework. The past century has seen constant improvements in this theory, with no less than two distinct Nobel prizes based on work that is essentially developments in VB theory. Additionally, ongoing advances in both methodology and computational power have greatly expanded the scope of problems that VB theory can address. In this Tutorial Review, we aim to give the reader a solid understanding of the foundations of modern VB theory, using a didactic example of a model S(N)2 reaction to illustrate its immediate applications. This will be complemented by examples of challenging problems that at present can only be efficiently addressed by VB-based approaches. Finally, the ongoing importance of VB theory is demonstrated. It is concluded that VB will continue to be a major driving force for chemistry in the century to come.

  • 176. Shyp, Viktoriya
    et al.
    Tankov, Stoyan
    Ermakov, Andrey
    Kudrin, Pavel
    English, Brian P
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tenson, Tanel
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Positive allosteric feedback regulation of the stringent response enzyme RelA by its product2012In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 13, no 9, p. 835-839Article in journal (Refereed)
    Abstract [en]

    During the stringent response, Escherichia coli enzyme RelA produces the ppGpp alarmone, which in turn regulates transcription, translation and replication. We show that ppGpp dramatically increases the turnover rate of its own ribosome-dependent synthesis by RelA, resulting in direct positive regulation of an enzyme by its product. Positive allosteric regulation therefore constitutes a new mechanism of enzyme activation. By integrating the output of individual RelA molecules and ppGpp degradation pathways, this regulatory circuit contributes to a fast and coordinated transition to stringency.

  • 177.
    Sooriyaarachchi, Sanjeewani
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Chofor, Rene
    Univ Ghent, Lab Med Chem FFW, Ottergemsesteenweg 460, B-9000 Ghent, Belgium..
    Risseeuw, Martijn D. P.
    Univ Ghent, Lab Med Chem FFW, Ottergemsesteenweg 460, B-9000 Ghent, Belgium..
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pouyez, Jenny
    Univ Namur, Dept Chem, Rue Bruxelles 61, B-5000 Namur, Belgium..
    Dowd, Cynthia S.
    George Washington Univ, Dept Chem, Washington, DC 20052 USA..
    Maes, Louis
    Univ Antwerp, LMPH, Univ Pl 1, B-2610 Antwerp, Belgium..
    Wouters, Johan
    Univ Namur, Dept Chem, Rue Bruxelles 61, B-5000 Namur, Belgium..
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Van Calenbergh, Serge
    Univ Ghent, Lab Med Chem FFW, Ottergemsesteenweg 460, B-9000 Ghent, Belgium..
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Targeting an Aromatic Hotspot in Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase with -Arylpropyl Analogues of Fosmidomycin2016In: ChemMedChem, ISSN 1860-7179, E-ISSN 1860-7187, Vol. 11, no 18, p. 2024-2036Article in journal (Refereed)
    Abstract [en]

    Blocking the 2-C-methyl-d-erythrithol-4-phosphate pathway for isoprenoid biosynthesis offers new ways to inhibit the growth of Plasmodium spp. Fosmidomycin [(3-(N-hydroxyformamido)propyl)phosphonic acid, 1] and its acetyl homologue FR-900098 [(3-(N-hydroxyacetamido)propyl)phosphonic acid, 2] potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this biosynthetic pathway. Arylpropyl substituents were introduced at the -position of the hydroxamate analogue of 2 to study changes in lipophilicity, as well as electronic and steric properties. The potency of several new compounds on the P.falciparum enzyme approaches that of 1 and 2. Activities against the enzyme and parasite correlate well, supporting the mode of action. Seven X-ray structures show that all of the new arylpropyl substituents displace a key tryptophan residue of the active-site flap, which had made favorable interactions with 1 and 2. Plasticity of the flap allows substituents to be accommodated in many ways; in most cases, the flap is largely disordered. Compounds can be separated into two classes based on whether the substituent on the aromatic ring is at the meta or para position. Generally, meta-substituted compounds are better inhibitors, and in both classes, smaller size is linked to better potency.

  • 178. Sooriyaarachchi, Sanjeewani
    et al.
    Jaber, Emad
    Covarrubias, Adrian Suarez
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ubhayasekera, Wimal
    Asiegbu, Frederick O.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Expression and beta-glucan binding properties of Scots pine (Pinus sylvestris L.) antimicrobial protein (Sp-AMP)2011In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 77, no 1-2, p. 33-45Article in journal (Refereed)
    Abstract [en]

    Scots pine (Pinus sylvestris) secretes a number of small, highly-related, disulfide-rich proteins (Sp-AMPs) in response to challenges with fungal pathogens such as Heterobasidion annosum, although their biological role has been unknown. Here, we examined the expression patterns of these genes, as well as the structure and function of the encoded proteins. Northern blots and quantitative real time PCR showed increased levels of expression that are sustained during the interactions of host trees with pathogens, but not non-pathogens, consistent with a function in conifer tree defenses. Furthermore, the genes were up-regulated after treatment with salicylic acid and an ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid, but neither methyl jasmonate nor H(2)O(2) induced expression, indicating that Sp-AMP gene expression is independent of the jasmonic acid signaling pathways. The cDNA encoding one of the proteins was cloned and expressed in Pichia pastoris. The purified protein had antifungal activity against H. annosum, and caused morphological changes in its hyphae and spores. It was directly shown to bind soluble and insoluble beta-(1,3)-glucans, specifically and with high affinity. Furthermore, addition of exogenous glucan is linked to higher levels of Sp-AMP expression in the conifer. Homology modeling and sequence comparisons suggest that a conserved patch on the surface of the globular Sp-AMP is a carbohydrate-binding site that can accommodate approximately four sugar units. We conclude that these proteins belong to a new family of antimicrobial proteins (PR-19) that are likely to act by binding the glucans that are a major component of fungal cell walls.

  • 179. Stern, Ana Laura
    et al.
    Naworyta, Agata
    Cazzulo, Juan J.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structures of type B ribose 5-phosphate isomerase from Trypanosoma cruzi shed light on the determinants of sugar specificity in the structural family2011In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 5, p. 793-808Article in journal (Refereed)
    Abstract [en]

    Ribose-5-phosphate isomerase (Rpi; EC 5.3.1.6) is a key activity of the pentose phosphate pathway. Two unrelated types of sequence/structure possess this activity: type A Rpi (present in most organisms) and type B Rpi (RpiB) (in some bacteria and parasitic protozoa). In the present study, we report enzyme kinetics and crystallographic studies of the RpiB from the human pathogen, Trypanosoma cruzi. Structures of the wild-type and a Cys69Ala mutant enzyme, alone or bound to phosphate, D-ribose 5-phosphate, or the inhibitors 4-phospho-D-erythronohydroxamic acid and D-allose 6-phosphate, highlight features of the active site, and show that small conformational changes are linked to binding. Kinetic studies confirm that, similar to the RpiB from Mycobacterium tuberculosis, the T. cruzi enzyme can isomerize D-ribose 5-phosphate effectively, but not the 6-carbon sugar D-allose 6-phosphate; instead, this sugar acts as an inhibitor of both enzymes. The behaviour is distinct from that of the more closely related (to T. cruzi RpiB) Escherichia coli enzyme, which can isomerize both types of sugars. The hypothesis that differences in a phosphate-binding loop near the active site were linked to the differences in specificity was tested by construction of a mutant T. cruzi enzyme with a sequence in this loop more similar to that of E. coli RpiB; this mutant enzyme gained the ability to act on the 6-carbon sugar. The combined information allows us to distinguish the two types of specificity patterns in other available sequences. The results obtained in the present study provide insights into the action of RpiB enzymes generally, and also comprise a firm basis for future work in drug design.

  • 180. Stojanoff, Vivian
    et al.
    Jakoncic, Jean
    Oren, Deena A.
    Nagarajan, V.
    Poulsen, Jens-Christian Navarro
    Adams-Cioaba, Melanie A.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sommer, Morten O. A.
    From screen to structure with a harvestable microfluidic device2011In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 67, p. 971-975Article in journal (Refereed)
    Abstract [en]

    Advances in automation have facilitated the widespread adoption of high-throughput vapour-diffusion methods for initial crystallization screening. However, for many proteins, screening thousands of crystallization conditions fails to yield crystals of sufficient quality for structural characterization. Here, the rates of crystal identification for thaumatin, catalase and myoglobin using microfluidic Crystal Former devices and sitting-drop vapour-diffusion plates are compared. It is shown that the Crystal Former results in a greater number of identified initial crystallization conditions compared with vapour diffusion. Furthermore, crystals of thaumatin and lysozyme obtained in the Crystal Former were used directly for structure determination both in situ and upon harvesting and cryocooling. On the basis of these results, a crystallization strategy is proposed that uses multiple methods with distinct kinetic trajectories through the protein phase diagram to increase the output of crystallization pipelines.

  • 181.
    Sun, Song
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Resistance to beta-Lactam Antibiotics Conferred by Point Mutations in Penicillin-Binding Proteins PBP3, PBP4 and PBP6 in Salmonella enterica2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 5, p. e97202-Article in journal (Refereed)
    Abstract [en]

    Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for beta-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a beta-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against beta-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against b-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became 'generalists" with increased resistance against several different classes of b-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to b-lactam antibiotics.

  • 182. Sun, Wei
    et al.
    Meng, Xiangyu
    Liang, Lingjie
    Jiang, Wangshu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Huang Almqvist, Yafei
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    He, Jing
    Hu, Haiyan
    Almqvist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gao, Xiang
    Wang, Li
    Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in Flavonoid Biosynthetic Pathway2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3Article in journal (Refereed)
    Abstract [en]

    Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

  • 183.
    Söderholm, Annika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Guo, Xiaohu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Newton, Matilda S.
    Evans, Gary B.
    Näsvall, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Patrick, Wayne M.
    Univ Otago, Dept Biochem, Dunedin 9054, New Zealand.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 41, p. 24657-24668Article in journal (Refereed)
    Abstract [en]

    HisA is a (βα)8 barrel enzyme that catalyzes the Amadori rearrangement of ProFAR to PRFAR in the histidine biosynthesis pathway and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp7 acts as the catalytic base and Asp176 as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure, and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen-bond network including His47, Asp129, Thr171 and Ser202, all shown to be functionally important. The closed-conformation structure is highly similar to the bi-functional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.

  • 184.
    Tek, Alex
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Chen, Yang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Flores, Samuel C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Investigating Ribosome Conformations with Multi-Resolution Modeling2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 491A-491AArticle in journal (Other academic)
  • 185. Tsai, Albert
    et al.
    Uemura, Sotaro
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Puglisi, Elisabetta Viani
    Marshall, R. Andrew
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Aitken, Colin Echeverria
    Korlach, Jonas
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Puglisi, Joseph D.
    The Impact of Aminoglycosides on the Dynamics of Translation Elongation2013In: Cell Reports, ISSN 2211-1247, Vol. 3, no 2, p. 497-508Article in journal (Refereed)
    Abstract [en]

    Inferring antibiotic mechanisms on translation through static structures has been challenging, as biological systems are highly dynamic. Dynamic single-molecule methods are also limited to few simultaneously measurable parameters. We have circumvented these limitations with a multifaceted approach to investigate three structurally distinct aminoglycosides that bind to the aminoacyl-transfer RNA site (A site) in the prokaryotic 30S ribosomal subunit: apramycin, paromomycin, and gentamicin. Using several single-molecule fluorescence measurements combined with structural and biochemical techniques, we observed distinct changes to translational dynamics for each aminoglycoside. While all three drugs effectively inhibit translation elongation, their actions are structurally and mechanistically distinct. Apramycin does not displace A1492 and A1493 at the decoding center, as demonstrated by a solution nuclear magnetic resonance structure, causing only limited miscoding; instead, it primarily blocks translocation. Paromomycin and gentamicin, which displace A1492 and A1493, cause significant miscoding, block intersubunit rotation, and inhibit translocation. Our results show the power of combined dynamics, structural, and biochemical approaches to elucidate the complex mechanisms underlying translation and its inhibition.

  • 186.
    Vovusha, Hakkim
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy.
    Banerjee, Debapriya
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Oumata, Nassima
    Sanyal, Biplab
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Electronic structure and spectroscopic properties of 6-aminophenanthridine and its derivatives: Insights from density functional theory2015In: International Journal of Quantum Chemistry, ISSN 0020-7608, E-ISSN 1097-461X, Vol. 115, no 13, p. 846-852Article in journal (Refereed)
    Abstract [en]

    6-Aminophenanthridine (6AP) and its derivatives show important biological activities as antiprion compounds and inhibitors of the protein folding activity of the ribosome. Both of these activities depend on the RNA binding property of these compounds, which has been recently characterized by fluorescence spectroscopy. Hence, fundamental insights into the photophysical properties of 6AP compounds are highly important to understand their biological activities. In this work, we have calculated electronic structures and optical properties of 6AP and its three derivatives 6AP8CF(3), 6AP8Cl, and 6APi by density functional theory (DFT) and time-dependent density functional theory (TDDFT). Our calculated spectra show a good agreement with the experimental absorption and fluorescence spectra, and thus, provide deep insights into the optical properties of the compounds. Furthermore, comparing the results obtained with four different hybrid functionals, we demonstrate that the accuracy of the functionals varies in the order B3LYP>PBE0>M062X>M06HF.

  • 187.
    Vovusha, Hakkim
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Biplab
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Adsorption of nucleobases on 2D transition-metal dichalcogenides and graphene sheet: a first principles density functional theory study2015In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 5, no 83, p. 67427-67434Article in journal (Refereed)
    Abstract [en]

    Adsorption characteristics of DNA/RNA nucleobases on monolayers of transition-metal dichalcogenide (TMD) such as molybdenum disulfide (MoS2) and tungsten disulfide (WS2) have been studied using first principles density functional theory (DFT) with vdW-DF method. The same calculations have been performed with PBE and DFT-D-2 method for comparison. In addition, a comparative study has been done for adsorption with graphene (GRA) also to compare with MoS2 and WS2. We have found that all nucleobases are physisorbed on MoS2 and WS2 due to van der Waals interaction, which is similar to that of nucleobases on GRA. The order of binding energy of the nucleobases with MoS2 and WS2 is G > A > T > C > U using vdW-DF and DFT-D-2 method, which is also similar to that of GRA-nucleobases. Without the inclusion of vdW interaction (PBE only), the order of the binding energies is calculated to be G > C > A > T > U for MoS2 and WS2-nucleobase complexes and G > C > A=T > U for GRA. We have analyzed changes in the electronic structures due to adsorption and the consequences in the calculated optical absorption spectra. Moreover, we have found that the calculated work functions of MoS2, WS2 and GRA decrease after the adsorption of nucleobases. Our results demonstrate that apart from graphene, transition metal dichalcogenides may also be used to detect biomolecules for medical science and biotechnology.