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  • 1.
    Amrein, Beat A.
    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.
    Bauer, Paul
    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.
    Duarte, Fernanda
    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.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Naworyta, Agata
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    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.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C. 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.
    Expanding the catalytic triad in epoxide hydrolases and related enzymes2015In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed)
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

  • 2.
    Amrein, Beat Anton
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Steffen-Munsberg, Fabian
    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.
    Szeler, Ireneusz
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Purg, Miha
    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.
    Kulkarni, Yashraj
    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.
    CADEE: Computer-Aided Directed Evolution of Enzymes2017In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 4, no 1, p. 50-64Article in journal (Refereed)
    Abstract [en]

    The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

  • 3.
    Andaloussi, Mounir
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Henriksson, Lena M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wieckowska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Lindh, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Larsson, Anna M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Suresh, Surisetti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Iyer, Harini
    Srinivasa, Bachally R.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure 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.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase2011In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 14, p. 4964-4976Article in journal (Refereed)
    Abstract [en]

    The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. alpha-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 mu M on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 mu g/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

  • 4.
    Andaloussi, Mounir
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Lindh, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Suresh, Surisetti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Wieckowska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Iyer, Harini
    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.
    Substitution of the phosphonic acid and hydroxamic acid functionalities of the DXR inhibitor FR900098: An attempt to improve the activity against Mycobacterium tuberculosis2011In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 21, no 18, p. 5403-5407Article in journal (Refereed)
    Abstract [en]

    Two series of FR900098/fosmidomycin analogs were synthesized and evaluated for MtDXR inhibition and Mycobacterium tuberculosis whole-cell activity. The design rationale of these compounds involved the exchange of either the phosphonic acid or the hydroxamic acid part for alternative acidic and metal-coordinating functionalities. The best inhibitors provided IC(50) values in the micromolar range, with a best value of 41 mu M.

  • 5. Andersson, Marlene
    et al.
    Chen, Gefei
    Otikovs, Martins
    Landreh, Michael
    Nordling, Kerstin
    Kronqvist, Nina
    Westermark, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Jornvall, Hans
    Knight, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ridderstrale, Yvonne
    Holm, Lena
    Meng, Qing
    Jaudzems, Kristaps
    Chesler, Mitchell
    Johansson, Jan
    Rising, Anna
    Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains2014In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 12, no 8, p. e1001921-Article in journal (Refereed)
    Abstract [en]

    Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive beta-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO(2)) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.

  • 6.
    Aqvist, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell 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.
    Exceptionally large entropy contributions enable the high rates of GTP hydrolysis on the ribosome2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 15817Article in journal (Refereed)
    Abstract [en]

    Protein synthesis on the ribosome involves hydrolysis of GTP in several key steps of the mRNA translation cycle. These steps are catalyzed by the translational GTPases of which elongation factor Tu (EF-Tu) is the fastest GTPase known. Here, we use extensive computer simulations to explore the origin of its remarkably high catalytic rate on the ribosome and show that it is made possible by a very large positive activation entropy. This entropy term (T Delta S-double dagger) amounts to more than 7 kcal/mol at 25 degrees C. It is further found to be characteristic of the reaction mechanism utilized by the translational, but not other, GTPases and it enables these enzymes to attain hydrolysis rates exceeding 500 s(-1). This entropy driven mechanism likely reflects the very high selection pressure on the speed of protein synthesis, which drives the rate of each individual GTPase towards maximal turnover rate of the whole translation cycle.

  • 7.
    Arand, M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. PROGRAM IN STRUCTURAL MOLECULAR BIOLOGY.
    Cronin, A
    Oesch, F
    Mowbray, Sherry L
    Department of Molecular Biosciences, Swedish University of Agricultural Sciences.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    The telltale structures of epoxide hydrolases2003In: Drug metabolism reviews (Softcover ed.), ISSN 0360-2532, E-ISSN 1097-9883, Vol. 35, no 4, p. 365-383Article in journal (Refereed)
    Abstract [en]

    Traditionally, epoxide hydrolases (EH) have been regarded as xenobiotic-metabolizing enzymes implicated in the detoxification of foreign compounds. They are known to play a key role in the control of potentially genotoxic epoxides that arise during metabolism of many lipophilic compounds. Although this is apparently the main function for the mammalian microsomal epoxide hydrolase (mEH), evidence is now accumulating that the mammalian soluble epoxide hydrolase (sEH), despite its proven role in xenobiotic metabolism, also has a central role in the formation and breakdown of physiological signaling molecules. In addition, a certain class of microbial epoxide hydrolases has recently been identified that is an integral part of a catabolic pathway, allowing the use of specific terpens as sole carbon sources. The recently available x-ray structures of a number of EHs mirror their respective functions: the microbial terpen EH differs in its fold from the canonical α/β hydrolase fold of the xenobiotic-metabolizing mammalian EHs. It appears that the latter fold is the perfect solution for the efficient detoxification of a large variety of structurally different epoxides by a single enzyme, whereas the smaller microbial EH, which has a particularly high turnover number with its prefered substrate, seems to be the better solution for the hydrolysis of one specific substrate. The structure of the sEH also includes an additional catalytic domain that has recently been shown to possess phosphatase activity. Although the physiological substrate for this second active site has not been identified so far, the majority of known phosphatases are involved in signaling processes, suggesting that the sEH phosphatase domain also has a role in the regulation of physiological functions.

  • 8.
    Artursson, Per
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Knight, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Breaking the intestinal barrier to deliver drugs2015In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 347, no 6223, p. 716-717Article in journal (Other academic)
  • 9.
    Banerjee, Debapriya
    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.
    Protein Folding Activity of the Ribosome (PFAR): A Target for Antiprion Compounds2014In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 6, no 10, p. 3907-3924Article, review/survey (Refereed)
    Abstract [en]

    Prion diseases are fatal neurodegenerative diseases affecting mammals. Prions are misfolded amyloid aggregates of the prion protein (PrP), which form when the alpha helical, soluble form of PrP converts to an aggregation-prone, beta sheet form. Thus, prions originate as protein folding problems. The discovery of yeast prion(s) and the development of a red-/white-colony based assay facilitated safe and high-throughput screening of antiprion compounds. With this assay three antiprion compounds; 6-aminophenanthridine (6AP), guanabenz acetate (GA), and imiquimod (IQ) have been identified. Biochemical and genetic studies reveal that these compounds target ribosomal RNA (rRNA) and inhibit specifically the protein folding activity of the ribosome (PFAR). The domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active site for PFAR. 6AP and GA inhibit PFAR by competition with the protein substrates for the common binding sites on the domain V rRNA. PFAR inhibition by these antiprion compounds opens up new possibilities for understanding prion formation, propagation and the role of the ribosome therein. In this review, we summarize and analyze the correlation between PFAR and prion processes using the antiprion compounds as tools.

  • 10.
    Banerjee, Debapriya
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Vovusha, Hakkim
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pang, Yanhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure 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.
    Spectroscopic and DFT studies on 6-Aminophenanthridine and its derivatives provide insights in their activity towards ribosomal RNA2014In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 97, p. 194-199Article in journal (Refereed)
    Abstract [en]

    6-Aminophenanthridine (6AP), a plant alkaloid possessing antiprion activity, inhibits ribosomal RNA dependent protein folding activity of the ribosome (referred as PFAR). We have compared 6AP and its three derivatives 6AP8Cl, 6AP8CF3 and 6APi for their activity in inhibition of PFAR. Since PFAR inhibition requires 6AP and its derivatives to bind to the ribosomal RNA (rRNA), we have measured the binding affinity of these molecules to domain V of 23S rRNA using fluorescence spectroscopy. Our results show that similar to the antiprion activity, both the inhibition of PFAR and the affinity towards rRNA follow the order 6AP8CF3 > 6AP8Cl > 6AP, while 6APi is totally inactive. To have a molecular insight for the difference in activity despite similarities in structure, we have calculated the nucleus independent chemical shift using first principles density functional theory. The result suggests that the deviation of planarity in 6APi and steric hindrance from its bulky side chain are the probable reasons which prevent it from interacting with rRNA. Finally, we suggest a probable mode of action of 6AP, 6AP8CF3 and 6AP8Cl towards rRNA.

  • 11.
    Barrozo, Alexandre
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Promiscuity and Selectivity in Phosphoryl Transferases2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Phosphoryl transfers are essential chemical reactions in key life processes, including energy production, signal transduction and protein synthesis. They are known for having extremely low reaction rates in aqueous solution, reaching the scale of millions of years. In order to make life possible, enzymes that catalyse phosphoryl transfer, phosphoryl transferases, have evolved to be tremendously proficient catalysts, increasing reaction rates to the millisecond timescale.

    Due to the nature of the electronic structure of phosphorus atoms, understanding how hydrolysis of phosphate esters occurs is a complex task. Experimental studies on the hydrolysis of phosphate monoesters with acidic leaving groups suggest a concerted mechanism with a loose, metaphosphate-like transition state. Theoretical studies have suggested two possible concerted pathways, either with loose or tight transition state geometries, plus the possibility of a stepwise mechanism with the formation of a phosphorane intermediate. Different pathways were shown to be energetically preferable depending on the acidity of the leaving group. Here we performed computational studies to revisit how this mechanistic shift occurs along a series of aryl phosphate monoesters, suggesting possible factors leading to such change.

    The fact that distinct pathways can occur in solution could mean that the same is possible for an enzyme active site. We performed simulations on the catalytic activity of β-phosphoglucomutase, suggesting that it is possible for two mechanisms to occur at the same time for the phosphoryl transfer.

    Curiously, several phosphoryl transferases were shown to be able to catalyse not only phosphate ester hydrolysis, but also the cleavage of other compounds. We modeled the catalytic mechanism of two highly promiscuous members of the alkaline phosphatase superfamily. Our model reproduces key experimental observables and shows that these enzymes are electrostatically flexible, employing the same set of residues to enhance the rates of different reactions, with different electrostatic contributions per residue.

    List of papers
    1. Evaluation and Characterisation of Mechanistic Alternatives for beta-Phosphoglucomutase
    Open this publication in new window or tab >>Evaluation and Characterisation of Mechanistic Alternatives for beta-Phosphoglucomutase
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-278943 (URN)
    Available from: 2016-02-26 Created: 2016-02-26 Last updated: 2016-04-12
    2. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Open this publication in new window or tab >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
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    2014 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 16, p. 4351-4362Article in journal (Refereed) Published
    Abstract [en]

    The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

    National Category
    Physical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
    Funder
    Swedish National Infrastructure for Computing (SNIC), 2013/26-1
    Available from: 2014-06-23 Created: 2014-06-04 Last updated: 2017-12-05
    3. Mechanistic Shifts Along the Linear Free Energy Relationship for Aryl Phosphate Monoester Hydrolysis
    Open this publication in new window or tab >>Mechanistic Shifts Along the Linear Free Energy Relationship for Aryl Phosphate Monoester Hydrolysis
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Theoretical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-278945 (URN)
    Available from: 2016-02-26 Created: 2016-02-26 Last updated: 2016-04-12
    4. Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily
    Open this publication in new window or tab >>Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily
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    2015 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, no 28, p. 9061-9076Article in journal (Refereed) Published
    Abstract [en]

    It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-260856 (URN)10.1021/jacs.5b03945 (DOI)000358556200033 ()26091851 (PubMedID)
    Funder
    Swedish Research Council, 2010-5026EU, FP7, Seventh Framework Programme, 306474Swedish National Infrastructure for Computing (SNIC), 25/2-10
    Note

    De 2 första författarna delar förstaförfattarskapet.

    Available from: 2015-08-26 Created: 2015-08-25 Last updated: 2017-12-04Bibliographically approved
  • 12.
    Barrozo, Alexandre
    et al.
    Univ Southern Calif, Dept Chem, Los Angeles, CA 90089 USA..
    Blaha-Nelson, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Williams, Nicholas H.
    Univ Sheffield, Dept Chem, Sheffield S3 7HF, S Yorkshire, England..
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    The effect of magnesium ions on triphosphate hydrolysis2017In: Pure and Applied Chemistry, ISSN 0033-4545, E-ISSN 1365-3075, Vol. 89, no 6, p. 715-727Article in journal (Refereed)
    Abstract [en]

    The role of metal ions in catalyzing phosphate ester hydrolysis has been the subject of much debate, both in terms of whether they change the transition state structure or mechanistic pathway. Understanding the impact of metal ions on these biologically critical reactions is central to improving our understanding of the role of metal ions in the numerous enzymes that facilitate them. In the present study, we have performed density functional theory studies of the mechanisms of methyl triphosphate and acetyl phosphate hydrolysis in aqueous solution to explore the competition between solvent-and substrate-assisted pathways, and examined the impact of Mg2+ on the energetics and transition state geometries. In both cases, we observe a clear preference for a more dissociative solvent-assisted transition state, which is not significantly changed by coordination of Mg2+. The effect of Mg2+ on the transition state geometries for the two pathways is minimal. While our calculations cannot rule out a substrate-assisted pathway as a possible solution for biological phosphate hydrolysis, they demonstrate that a significantly higher energy barrier needs to be overcome in the enzymatic reaction for this to be an energetically viable reaction pathway.

  • 13.
    Barrozo, Alexandre
    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.
    Duarte, Fernanda
    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.
    Bauer, Paul
    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.
    Carvalho, Alexandra T. P.
    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 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.
    Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily2015In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, no 28, p. 9061-9076Article in journal (Refereed)
    Abstract [en]

    It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.

  • 14.
    Barrozo, Alexandre
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Esguerra, Mauricio
    Marloie, Gael
    Florian, Jan
    Williams, Nicholas
    Kamerlin, Shina
    Evaluation and Characterisation of Mechanistic Alternatives for beta-PhosphoglucomutaseManuscript (preprint) (Other academic)
  • 15.
    Barrozo, Alexandre
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Brandao, Tiago
    Hengge, Alvan
    Phosphoryl and Sulfuryl Transfer2016In: Reference Module in Chemistry, Molecular Sciences and Chemical EngineeringArticle in journal (Refereed)
  • 16.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Computational modelling of enzyme selectivity2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enantioselective reactions are one of the ways to produce pure chiral compounds. Understanding the basis of this selectivity makes it possible to guide enzyme design towards more efficient catalysts. One approach to study enzymes involved in chiral chemistry is through the use of computational models that are able to simulate the chemical reaction taking place. The potato epoxide hydrolase is one enzyme that is known to be both highly enantioselective, while still being robust upon mutation of residues to change substrate scope. The enzyme was used to investigate the epoxide hydrolysis mechanism for a number of different substrates, using the EVB approach to the reaction both in solution and in several enzyme variants. In addition to this, work has been performed on new ways of performing simulations of divalent transition metals, as well as development of new simulation software.

    List of papers
    1. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Open this publication in new window or tab >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
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    2014 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 16, p. 4351-4362Article in journal (Refereed) Published
    Abstract [en]

    The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

    National Category
    Physical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
    Funder
    Swedish National Infrastructure for Computing (SNIC), 2013/26-1
    Available from: 2014-06-23 Created: 2014-06-04 Last updated: 2017-12-05
    2. Expanding the catalytic triad in epoxide hydrolases and related enzymes
    Open this publication in new window or tab >>Expanding the catalytic triad in epoxide hydrolases and related enzymes
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    2015 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed) Published
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-260232 (URN)10.1021/acscatal.5b01639 (DOI)000362391500006 ()
    Funder
    EU, FP7, Seventh Framework Programme, 306474Swedish Research Council, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12
    Available from: 2015-08-18 Created: 2015-08-18 Last updated: 2017-12-04Bibliographically approved
    3. Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
    Open this publication in new window or tab >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
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    2016 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed) Published
    Abstract [en]

    Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
    Funder
    Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, European Research Council, 306474;283570Swedish Research Council, 621-2011-6055Carl Tryggers foundation , CTS13:104
    Available from: 2016-04-01 Created: 2016-04-01 Last updated: 2017-11-30Bibliographically approved
    4. Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis
    Open this publication in new window or tab >>Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis
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    2016 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 18, p. 1693-1697Article in journal (Refereed) Published
    Abstract [en]

    Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)-3-phenylpropane-1,2-diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)-diol is not only due to changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)-benzyloxirane. To probe the structural origin of these differences in substrate selectivities and catalytic regiopreferences, we have solved the crystal structures for the in-vitro evolved StEH1 variants. We have additionally used these structures as a starting point for docking the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking calculations, the apparent preferred binding modes obtained from the docking appears to rationalize the experimentally determined regioselectivities. These calculations could also identify an active site residue (F33) as a putatively important interaction partner, a role that could explain the high degree of conservation of this residue during evolution. Overall, our combined experimental, structural and computational studies of this system provide snapshots into the evolution of enantioconvergence in StEH1 catalyzed epoxide hydrolysis.

    Keyword
    enantioselectivity; epoxide hydrolysis; evolutionary snapshots; laboratory evolution; protein engineering
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-298675 (URN)10.1002/cbic.201600330 (DOI)000384425400004 ()27383542 (PubMedID)
    Funder
    Swedish Research CouncilEU, European Research Council, 306474Swedish National Infrastructure for Computing (SNIC), SNIC2015-16-12EU, FP7, Seventh Framework Programme, 283570
    Available from: 2016-07-06 Created: 2016-07-06 Last updated: 2017-11-28Bibliographically approved
    5. Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
    Open this publication in new window or tab >>Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
    Show others...
    (English)In: Article in journal (Other academic) Submitted
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-325485 (URN)
    Available from: 2017-07-02 Created: 2017-07-02 Last updated: 2018-03-23
    6. Q Version 6, a comprehensive toolkit for empirical valence bond and related free energy calculations.
    Open this publication in new window or tab >>Q Version 6, a comprehensive toolkit for empirical valence bond and related free energy calculations.
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Other Chemistry Topics
    Identifiers
    urn:nbn:se:uu:diva-325490 (URN)
    Available from: 2017-07-02 Created: 2017-07-02 Last updated: 2017-07-03
  • 17.
    Bauer, Paul
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Barrozo, Alexandre
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Amrein, Beat Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Purg, Miha
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Esguerra, Mauricio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Wilson, Philippe
    De Montfort University Leicester, School of Pharmacy .
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Major, Dan Thomas
    Department of Chemistry, The Lise Meitner-Minerva Center of Computational Quantum Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Q Version 6, a comprehensive toolkit for empirical valence bond and related free energy calculations.Manuscript (preprint) (Other academic)
  • 18.
    Bauer, Paul
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Amrein, Beat A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, S. C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 12016In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed)
    Abstract [en]

    Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

  • 19. Ben-David, Moshe
    et al.
    Sussman, Joel L.
    Maxwel, Christopher L.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Szeler, Klaudia
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Lynn Shina C.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Tawfik, Dan S.
    Catalytic Stimulation by Restrained Active-Site Floppiness-The Case of High Density Lipoprotein-Bound Serum Paraoxonase-12015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 6, p. 1359-1374Article in journal (Refereed)
    Abstract [en]

    Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168-a key catalytic residue residing >15 angstrom from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.

  • 20.
    Björkelid, Christofer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Henriksson, Lena M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Stern, Ana Laura
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure 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.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structural and functional studies of mycobacterial IspD enzymes2011In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 67, p. 403-414Article in journal (Refereed)
    Abstract [en]

    A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.

  • 21.
    Björkelid, Christofer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Raichurkar, Anand Kumar V.
    AstraZeneca India Private Limited.
    Mukherjee, Kakoli
    AstraZeneca India Private Limited.
    Malolanarasimhan, Krishnan
    AstraZeneca India Private Limited.
    Bandodkar, Balachandra
    AstraZeneca India Private Limited.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structural and biochemical characterization of compounds inhibiting Mycobacterium tuberculosis Pantothenate Kinase2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 25, p. 18260-18270Article in journal (Refereed)
    Abstract [en]

    Mycobacterium tuberculosis, the bacterial causative agent oftuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemicalcharacterizations of two new classes of compounds that inhibitpantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenateand phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for anypantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.

  • 22.
    Björkelid, Christofer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure 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.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-9000982012In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 68, p. 134-143Article in journal (Refereed)
    Abstract [en]

    A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity.

  • 23.
    Blaha-Nelson, David
    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.
    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.
    Szeler, Klaudia
    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.
    Ben-David, Moshe
    Weizmann Inst Sci, Dept Biol Chem, IL-76100 Rehovot, Israel.;Univ Toronto, Banting & Best Dept Med Res, Donnelly Ctr Cellular & Biomol Res, 160 Coll St, Toronto, ON M5S 3E1, Canada..
    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.
    Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 12017In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 139, no 3, p. 1155-1167Article in journal (Refereed)
    Abstract [en]

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed beta-propeller with a flexible loop (residues 70-81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1's lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1's lactonase activity is minimal, whereas the k(cat) for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1's active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar "gating loop" or a highly buried solvent excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates.

  • 24.
    Borg, Anneli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mechanisms and Inhibition of EF-G-dependent Translocation and Recycling of the Bacterial Ribosome2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The GTPase elongation factor G (EF-G) is an important player in the complex process of protein synthesis by bacterial ribosomes. Although extensively studied much remains to be learned about this fascinating protein. In the elongation phase, after incorporation of each amino acid into the growing peptide chain, EF-G translocates the ribosome along the mRNA template. In the recycling phase, when the synthesis of a protein has been completed, EF-G, together with ribosome recycling factor (RRF), splits the ribosome into its subunits. We developed the first in vitro assay for measuring the average time of a complete translocation step at any position along the mRNA. Inside the open reading frame, at saturating EF-G concentration and low magnesium ion concentration, translocation rates were fast and compatible with elongation rates observed in vivo. We also determined the complete kinetic mechanism for EF-G- and RRF-dependent splitting of the post-termination ribosome. We showed that splitting occurs only when RRF binds before EF-G and that the rate and GTP consumption of the reaction varies greatly with the factor concentrations.

    The antibiotic fusidic acid (FA) inhibits bacterial protein synthesis by binding to EF-G when the factor is ribosome bound, during translocation and ribosome recycling. We developed experimental methods and a theoretical framework for analyzing the effect of tight-binding inhibitors like FA on protein synthesis. We found that FA targets three different states during each elongation cycle and that it binds to EF-G on the post-termination ribosome both in the presence and absence of RRF. The stalling time of an FA-inhibited ribosome is about hundred-fold longer than the time of an uninhibited elongation cycle and therefore each binding event has a large impact on the protein synthesis rate and may induce queuing of ribosomes on the mRNA. Although ribosomes in the elongation and the recycling phases are targeted with similar efficiency, we showed that the main effect of FA in vivo is on elongation. Our results may serve as a basis for modelling of EF-G function and FA inhibition inside the living cell and for structure determination of mechanistically important intermediate states in translocation and ribosome recycling.

    List of papers
    1. Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis
    Open this publication in new window or tab >>Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis
    2015 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 9, p. 1835-1847Article in journal (Refereed) Published
    Abstract [en]

    Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30 s(-1) at 1 mM free Mg2+ concentration to the detrimentally low value of 1 s(-1) at 6 mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0-6 kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account. (C) 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-255072 (URN)10.1016/j.jmb.2014.10.027 (DOI)000353929400005 ()25451025 (PubMedID)
    Available from: 2015-06-15 Created: 2015-06-12 Last updated: 2018-01-11Bibliographically approved
    2. Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome
    Open this publication in new window or tab >>Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome
    Show others...
    2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, p. 3440-3454Article in journal (Refereed) Published
    Abstract [en]

    The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation(III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K-50% approximate to 1 mu M), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-247496 (URN)10.1074/jbc.M114.611608 (DOI)000349456000020 ()25451927 (PubMedID)
    Available from: 2015-03-19 Created: 2015-03-19 Last updated: 2018-01-11Bibliographically approved
    3. Complete kinetic mechanism for recycling of the bacterial ribosome
    Open this publication in new window or tab >>Complete kinetic mechanism for recycling of the bacterial ribosome
    2016 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 22, no 1, p. 10-21Article in journal (Refereed) Published
    Abstract [en]

    How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of 5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.

    Keyword
    bacterial ribosome recycling; elongation factor G; ribosome recycling factor; translation rate optimization; protein synthesis
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-258988 (URN)10.1261/rna.053157.115 (DOI)000368967600002 ()26527791 (PubMedID)
    Funder
    Swedish Research CouncilKnut and Alice Wallenberg Foundation
    Available from: 2015-08-04 Created: 2015-07-23 Last updated: 2017-12-04Bibliographically approved
    4. Fusidic acid inhibition of EF-G- and RRF-promoted recycling of the bacterial ribosome
    Open this publication in new window or tab >>Fusidic acid inhibition of EF-G- and RRF-promoted recycling of the bacterial ribosome
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-258989 (URN)
    Available from: 2015-08-04 Created: 2015-07-23 Last updated: 2015-10-01
  • 25.
    Borg, Anneli
    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.
    Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 9, p. 1835-1847Article in journal (Refereed)
    Abstract [en]

    Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30 s(-1) at 1 mM free Mg2+ concentration to the detrimentally low value of 1 s(-1) at 6 mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0-6 kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account. (C) 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

  • 26.
    Borg, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Holm, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Shiroyama, Ikue
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael
    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.
    Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, p. 3440-3454Article in journal (Refereed)
    Abstract [en]

    The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation(III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K-50% approximate to 1 mu M), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

  • 27.
    Borg, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael
    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.
    Complete kinetic mechanism for recycling of the bacterial ribosome2016In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 22, no 1, p. 10-21Article in journal (Refereed)
    Abstract [en]

    How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of 5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.

  • 28.
    Borg, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael
    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.
    Fusidic acid inhibition of EF-G- and RRF-promoted recycling of the bacterial ribosomeManuscript (preprint) (Other academic)
  • 29.
    Borg, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael
    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.
    Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 7, p. 3264-3275Article in journal (Refereed)
    Abstract [en]

    The antibiotic drug fusidic acid (FA) is commonly used in the clinic against gram-positive bacterial infections. FA targets ribosome-bound elongation factor G (EF-G), a translational GTPase that accelerates both messenger RNA (mRNA) translocation and ribosome recycling. How FA inhibits translocation was recently clarified, but FA inhibition of ribosome recycling by EF-G and ribosome recycling factor (RRF) has remained obscure. Here we use fast kinetics techniques to estimate mean times of ribosome splitting and the stoichiometry of GTP hydrolysis by EF-G at varying concentrations of FA, EF-G and RRF. These mean times together with previous data on uninhibited ribosome recycling were used to clarify the mechanism of FA inhibition of ribosome splitting. The biochemical data on FA inhibition of translocation and recycling were used to model the growth inhibitory effect of FA on bacterial populations. We conclude that FA inhibition of translocation provides the dominant cause of bacterial growth reduction, but that FA inhibition of ribosome recycling may contribute significantly to FA-induced expression of short regulatory open reading frames, like those involved in FA resistance.

  • 30.
    Brandis, Gerrit
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wrande, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Liljas, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fitness-compensatory mutations in rifampicin-resistant RNA polymerase2012In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 85, no 1, p. 142-151Article in journal (Refereed)
    Abstract [en]

    Mutations in rpoB (RNA polymerase beta-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (RifR) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized RifR mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a RifR mutant. 

  • 31.
    Carvalho, Alexandra T. P.
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gouveia, Leonor
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Kanna, Charan Raju
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Warmlander, Sebastian K. T. S.
    Platts, Jamie A.
    Kamerlin, Lynn Shina Caroline
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Understanding the structural and dynamic consequences of DNA epigenetic modifications: Computational insights into cytosine methylation and hydroxymethylation2014In: Epigenetics, ISSN 1559-2294, E-ISSN 1559-2308, Vol. 9, no 12, p. 1604-1612Article in journal (Refereed)
    Abstract [en]

    We report a series of molecular dynamics (MD) simulations of up to a microsecond combined simulation time designed to probe epigenetically modified DNA sequences. More specifically, by monitoring the effects of methylation and hydroxymethylation of cytosine in different DNA sequences, we show, for the first time, that DNA epigenetic modifications change the molecule's dynamical landscape, increasing the propensity of DNA toward different values of twist and/or roll/tilt angles (in relation to the unmodified DNA) at the modification sites. Moreover, both the extent and position of different modifications have significant effects on the amount of structural variation observed. We propose that these conformational differences, which are dependent on the sequence environment, can provide specificity for protein binding.

  • 32.
    Carvalho, Alexandra T. P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Szeler, Klaudia
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Vavitsas, Konstantinos
    Univ Copenhagen, Dept Plant & Environm Sci, CPSC, DK-1871 Frederiksberg C, Denmark..
    Åqvist, 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.
    Kamerlin, Lynn Shina C.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Modeling the mechanisms of biological GTP hydrolysis2015In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 582, no SI, p. 80-90Article, review/survey (Refereed)
    Abstract [en]

    Enzymes that hydrolyze GTP are currently in the spotlight, due to their molecular switch mechanism that controls many cellular processes. One of the best-known classes of these enzymes are small GTPases such as members of the Ras superfamily, which catalyze the hydrolysis of the gamma-phosphate bond in GTP. In addition, the availability of an increasing number of crystal structures of translational GTPases such as EF-Tu and EF-G have made it possible to probe the molecular details of GTP hydrolysis on the ribosome. However, despite a wealth of biochemical, structural and computational data, the way in which GTP hydrolysis is activated and regulated is still a controversial topic and well-designed simulations can play an important role in resolving and rationalizing the experimental data. In this review, we discuss the contributions of computational biology to our understanding of GTP hydrolysis on the ribosome and in small GTPases.

  • 33.
    Chai, Qian
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Singh, Bhupender
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Peisker, Kristin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Metzendorf, Nicole
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ge, Xueliang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence2014In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 16, p. 11342-11352Article in journal (Refereed)
    Abstract [en]

    Background: We studied ribosome and nucleoid distribution in Escherichia coli under growth and quiescence. Results: Spatially segregated ribosomes and nucleoids show drastically altered distribution in stationary phase or when treated with drugs affecting translation, transcription, nucleoid-topology, or cytoskeleton. Ribosome inheritance in daughter cells is frequently unequal. Conclusion: Cellular growth processes modulate ribosome and nucleoid distribution. Significance: This provides insight into subcellular organization of molecular machines. We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.

  • 34.
    Chan, Sherwin
    et al.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden..
    Frasch, Alejandra
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden..
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ch'ng, Jun-Hong
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden.;Natl Univ Singapore, Dept Microbiol, Singapore 117545, Singapore..
    del Pilar Quintana, Maria
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden.;Univ Rosario, Fac Ciencias Nat & Matemat, Escuela Med & Ciencias Salud, Calle 12C 6-25, Bogota, Colombia..
    Vesterlund, Mattias
    Karolinska Inst, Dept Oncol Pathol, Canc Prote, S-17176 Stockholm, Sweden..
    Ghorbal, Mehdi
    Univ Montpellier, Lab Parasitol Mycol, Fac Med, F-34090 Montpellier, France.;Univ Montpellier, UMR MiVEGEC, IRD 224, CNRS 5290, Montpellier, France..
    Joannin, Nicolas
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden..
    Franzen, Oscar
    Icahn Sch Med Mt Sinai, Inst Genom & Multiscale Biol, Dept Genet & Genom Sci, New York, NY 10029 USA..
    Lopez-Rubio, Jose-Juan
    Univ Montpellier, Lab Parasitol Mycol, Fac Med, F-34090 Montpellier, France.;Univ Montpellier, UMR MiVEGEC, IRD 224, CNRS 5290, Montpellier, France..
    Barbieri, Sonia
    Univ Svizzera Italiana, Inst Res Biomed, CH-6500 Bellinzona, Switzerland..
    Lanzavecchia, Antonio
    Univ Svizzera Italiana, Inst Res Biomed, CH-6500 Bellinzona, Switzerland.;ETH, Inst Microbiol, CH-8093 Zurich, Switzerland..
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wahlgren, Mats
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden..
    Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor2017In: Nature Microbiology, E-ISSN 2058-5276, Vol. 2, no 7, article id 17068Article in journal (Refereed)
    Abstract [en]

    Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.

  • 35.
    Chen, Hongxin
    et al.
    Univ Helsinki, Dept Forest Sci, FIN-00014 Helsinki, Finland..
    Quintana, Julia
    Univ Helsinki, Dept Forest Sci, FIN-00014 Helsinki, Finland..
    Kovalchuk, Andriy
    Univ Helsinki, Dept Forest Sci, FIN-00014 Helsinki, Finland..
    Ubhayasekera, Wimal
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Asiegbu, Fred O.
    Univ Helsinki, Dept Forest Sci, FIN-00014 Helsinki, Finland..
    A cerato-platanin-like protein HaCPL2 from Heterobasidion annosum sensu stricto induces cell death in Nicotiana tabacum and Pinus sylvestris2015In: Fungal Genetics and Biology, ISSN 1087-1845, E-ISSN 1096-0937, Vol. 84, p. 41-51Article in journal (Refereed)
    Abstract [en]

    The cerato-platanin family is a group of small secreted cysteine-rich proteins exclusive for filamentous fungi. They have been shown to be involved in the interactions between fungi and plants. Functional characterization of members from this family has been performed mainly in Ascomycota, except Moniliophthora perniciosa. Our previous phylogenetic analysis revealed that recent gene duplication of cerato-platanins has occurred in Basidiomycota but not in Ascomycota, suggesting higher functional diversification of this protein family in Basidiomycota than in Ascomycota. In this study, we identified three cerato-platanin homologues from the basidiomycete conifer pathogen Heterobasidion annosum sensu stricto. Expression of the homologues under various conditions as well as their roles in the H. annosum s.s.-Pinus sylvestris (Scots pine) pathosystem was investigated. Results showed that HaCPL2 (ceratoplatanin-like protein 2) had the highest sequence similarity to cerato-platanin from Ceratocystis platani and hacpl2 was significantly induced during nutrient starvation and necrotrophic growth. The treatment with recombinant HaCPL2 induced cell death, phytoalexin production and defense gene expression in Nicotiana tabacum. Eliciting and cell death-inducing ability accompanied by retardation of apical root growth was also demonstrated in Scots pine seedlings. Our results suggest that HaCPL2 might contribute to the virulence of H. annosum s.s. by promoting plant cell death.

  • 36.
    Chen, Yang
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Näsvall, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wu, Shiying
    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.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3'')(9) adenyltransferase2015In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 71, p. 2267-2277Article in journal (Refereed)
    Abstract [en]

    Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleo\-tidyltransferases (ANTs). Here, the first crystal structure of an ANT(3$^\prime$$^\prime$)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

  • 37. Chofor, Rene
    et al.
    Sooriyaarachchi, Sanjeewani
    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.
    Risseeuw, Martijn D. P.
    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
    Johny, Chinchu
    Haymond, Amanda
    Everaert, Annelien
    Dowd, Cynthia S.
    Maes, Louis
    Coenye, Tom
    Alex, Alexander
    Couch, Robin D.
    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.
    Wouters, Johan
    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.
    Van Calenbergh, Serge
    Synthesis and Bioactivity of beta-Substituted Fosmidomycin Analogues Targeting 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase2015In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 58, no 7, p. 2988-3001Article in journal (Refereed)
    Abstract [en]

    Blocking the 2-C-methyl-d-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the beta-position of the hydroxamate analogue of 2. While direct addition of a beta-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the longer compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamates methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth.

  • 38.
    Choi, Junhong
    et al.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Dept Appl Phys, Stanford, CA 94305 USA..
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Demirci, Hasan
    SLAC Natl Accelerator Lab, Stanford PULSE Inst, Menlo Pk, CA USA.;SLAC Natl Accelerator Lab, Stanford Synchrotron Radiat Lightsource, Menlo Pk, CA USA..
    Chen, Jin
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Dept Appl Phys, Stanford, CA 94305 USA..
    Petrov, Alexey
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    Prabhakar, Arjun
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Program Biophys, Stanford, CA 94305 USA..
    O'Leary, Sean E.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    Dominissini, Dan
    Chaim Sheba Med Ctr, Canc Res Ctr, IL-52621 Tel Hashomer, Israel.;Univ Chicago, Dept Chem, 5735 S Ellis Ave, Chicago, IL 60637 USA..
    Rechavi, Gideon
    Chaim Sheba Med Ctr, Canc Res Ctr, IL-52621 Tel Hashomer, Israel.;Tel Aviv Univ, Israel & Sackler Sch Med, IL-69978 Tel Aviv, Israel..
    Soltis, S. Michael
    SLAC Natl Accelerator Lab, Stanford Synchrotron Radiat Lightsource, Menlo Pk, CA USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Puglisi, Joseph D.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    N-6-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics2016In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 23, no 2, p. 110-+Article in journal (Refereed)
    Abstract [en]

    N-6-methylation of adenosine (forming m(6)A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m(6)A in mRNA decoding. Although m(6)A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m(6)A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m(6)A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m(6)A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.

  • 39.
    Cuetos, Anibal
    et al.
    Univ York, York Struct Biol Lab, York YO10 5DD, N Yorkshire, England..
    Steffen-Munsberg, Fabian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanchez, Juan Mangas
    Univ York, York Struct Biol Lab, York YO10 5DD, N Yorkshire, England..
    Frese, Amina
    Univ York, York Struct Biol Lab, York YO10 5DD, N Yorkshire, England..
    Bornscheuer, Uwe T.
    Ernst Moritz Arndt Univ Greifswald, Inst Biochem, Felix Hausdorff Str 4, D-17487 Greifswald, Germany..
    Hoehne, Matthias
    Ernst Moritz Arndt Univ Greifswald, Inst Biochem, Felix Hausdorff Str 4, D-17487 Greifswald, Germany..
    Grogan, Gideon
    Univ York, York Struct Biol Lab, York YO10 5DD, N Yorkshire, England..
    Structural Basis for Phospholyase Activity of a Class III Transaminase Homologue2016In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 24, p. 2308-2311Article in journal (Refereed)
    Abstract [en]

    Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a remarkable diversity of chemical reactions in nature. A1RDF1 from Arthrobacter aurescens TC1 is a fold type I, PLP-dependent enzyme in the class III transaminase (TA) subgroup. Despite sharing 28% sequence identity with its closest structural homologues, including beta-alanine: pyruvate and gamma-aminobutyrate: alpha-ketoglutarate TAs, A1RDF1 displayed no TA activity. Activity screening revealed that the enzyme possesses phospholyase (E.C. 4.2.3.2) activity towards O-phosphoethanolamine (PEtN), an activity described previously for vertebrate enzymes such as human AGXT2L1, enzymes for which no structure has yet been reported. In order to shed light on the distinctive features of PLP-dependent phospholyases, structures of A1RDF1 in complex with PLP (internal aldimine) and PLP.PEtN (external aldimine) were determined, revealing the basis of substrate binding and the structural factors that distinguish the enzyme from class III homologues that display TA activity.

  • 40. D'Arcy, Allan
    et al.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Cowan-Jacob, Sandra W.
    Marsh, May
    Microseed matrix screening for optimization in protein crystallization: what have we learned?2014In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 70, p. 1117-1126Article in journal (Refereed)
    Abstract [en]

    Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time-and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.

  • 41.
    De Rosa, Maria
    et al.
    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.
    Lu, Lu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Zamaratski, Edouard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Szałaj, Natalia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wadensten, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lenhammar, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal 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.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Karlen, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)2017In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 3, p. 897-911Article in journal (Refereed)
    Abstract [en]

    Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.

  • 42.
    Degiacomi, Giulia
    et al.
    Univ Padua, Dept Mol Med, Via Gabelli 63, I-35121 Padua, Italy..
    Personne, Yoann
    Queen Mary Univ London, London E1 2AD, England.;UCL, Div Infect & Immun, Ctr Clin Microbiol, London, England..
    Mondesert, Guillaume
    Sanofi Aventis R&D, Drug Disposit, F-69367 Lyon, France..
    Ge, Xueliang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Hartkoorn, Ruben C.
    Ecole Polytech Fed Lausanne, Global Hlth Inst, Lausanne, Switzerland.;Univ Lille Nord France, Inst Pasteur Lille, Ctr Infect & Immun Lille, INSERM,CNRS,UMR 8204,U1019, Lille, France..
    Boldrin, Francesca
    Univ Padua, Dept Mol Med, Via Gabelli 63, I-35121 Padua, Italy..
    Goel, Pavitra
    Queen Mary Univ London, London E1 2AD, England..
    Peisker, Kristin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Benjak, Andrej
    Ecole Polytech Fed Lausanne, Global Hlth Inst, Lausanne, Switzerland..
    Barrio, Maria Belen
    Sanofi Aventis R&D, Drug Disposit, F-69367 Lyon, France..
    Ventura, Marcello
    Univ Padua, Dept Mol Med, Via Gabelli 63, I-35121 Padua, Italy..
    Brown, Amanda C.
    Queen Mary Univ London, London E1 2AD, England.;Cornell Univ, Dept Microbiol & Immunol, Ithaca, NY 14853 USA..
    Leblanc, Veronique
    Sanofi Aventis R&D, Drug Disposit, F-69367 Lyon, France..
    Bauer, Armin
    Sanofi Aventis R&D, Drug Disposit, F-69367 Lyon, France..
    Sanyal, Suparna
    Cole, Stewart T.
    Ecole Polytech Fed Lausanne, Global Hlth Inst, Lausanne, Switzerland..
    Lagrange, Sophie
    Sanofi Aventis R&D, Drug Disposit, F-69367 Lyon, France..
    Parish, Tanya
    Queen Mary Univ London, London E1 2AD, England.;Infect Dis Res Inst, TB Discovery Res, Seattle, WA 98102 USA..
    Manganelli, Riccardo
    Univ Padua, Dept Mol Med, Via Gabelli 63, I-35121 Padua, Italy..
    Micrococcin P1-A bactericidal thiopeptide active against Mycobacterium tuberculosis2016In: Tuberculosis, ISSN 1472-9792, E-ISSN 1873-281X, Vol. 100, p. 95-101Article in journal (Refereed)
    Abstract [en]

    The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains. In addition, in the case of TB, which claimed 1.5 million lives in 2014, the treatment of the least complicated, drug sensitive cases is lengthy and disagreeable. Therefore, new drugs with novel targets are urgently needed to control resistant Mycobacterium tuberculosis strains. In this manuscript we report the characterization of the thiopeptide micrococcin P1 as an anti-tubercular agent. Our biochemical experiments show that this antibiotic inhibits the elongation step of protein synthesis in mycobacteria. We have further identified micrococcin resistant mutations in the ribosomal protein L11 (RplK); the mutations were located in the proline loop at the N-terminus. Reintroduction of the mutations into a clean genetic background, confirmed that they conferred resistance, while introduction of the wild type RplK allele into resistant strains re-established sensitivity. We also identified a mutation in the 23S rRNA gene. These data, in good agreement with previous structural studies suggest that also in M. tuberculosis micrococcin P1 functions by binding to the cleft between the 23S rRNA and the L11 protein loop, thus interfering with the binding of elongation factors Tu and G (EF-Tu and EF-G) and inhibiting protein translocation.

  • 43. Deroo, Stephanie
    et al.
    Hyung, Suk-Joon
    Marcoux, Julien
    Gordiyenko, Yuliya
    Koripella, Ravi Kiran
    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.
    Robinson, Carol V.
    Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G2012In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 7, no 6, p. 1120-1127Article in journal (Refereed)
    Abstract [en]

    The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. On the basis of kinetic analysis, we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by similar to 27% and of dimers by similar to 47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.

  • 44.
    Di Yu, Xiao
    et al.
    Swedish Univ Agr Sci, Dept Mol Biol, Uppsala Bioctr, BMC, SE-75324 Uppsala, Sweden..
    Fooks, Laura J.
    Univ Reading, Sch Biol Sci, Reading RG6 6AJ, Berks, England..
    Moslehi-Mohebi, Elham
    Univ Reading, Sch Biol Sci, Reading RG6 6AJ, Berks, England..
    Tischenko, Vladimir M.
    Inst Biol Instrumentat, Pushchino 142292, Russia..
    Askarieh, Gelareh
    Swedish Univ Agr Sci, Dept Mol Biol, Uppsala Bioctr, BMC, SE-75324 Uppsala, Sweden..
    Knight, Stefan D.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Swedish Univ Agr Sci, Dept Mol Biol, Uppsala Bioctr, BMC, SE-75324 Uppsala, Sweden..
    MacIntyre, Sheila
    Univ Reading, Sch Biol Sci, Reading RG6 6AJ, Berks, England..
    Zavialov, Anton V.
    Swedish Univ Agr Sci, Dept Mol Biol, Uppsala Bioctr, BMC, SE-75324 Uppsala, Sweden.;Univ Turku, Dept Chem, FIN-20520 Turku, Finland..
    Large Is Fast, Small Is Tight: Determinants of Speed and Affinity in Subunit Capture by a Periplasmic Chaperone2012In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 417, no 4, p. 294-308Article in journal (Refereed)
    Abstract [en]

    The chaperone/usher pathway assembles surface virulence organelles of Gram-negative bacteria, consisting of fibers of linearly polymerized protein subunits. Fiber subunits are connected through 'donor strand complementation': each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh beta-strand in trans. Whereas the folding of Ig domains is a fast first-order process, folding of Ig modules into the fiber conformation is a slow second-order process. Periplasmic chaperones separate this process in two parts by forming transient complexes with subunits. Interactions between chaperones and subunits are also based on the principle of donor strand complementation. In this study, we have performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 capsular subunit from Yersinia pestis and analyzed the effect of the mutations on the structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. The results suggest that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enables Caf1M to rapidly bind an early folding intermediate of Caf1 and direct its partial folding. The switch from the Caf1M-Caf1 contact to the less hydrophobic, but considerably tighter and less dynamic Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M was engineered to bind Caf1 faster, tighter, or both faster and tighter. To our knowledge, this is the first successful attempt to rationally design an assembly chaperone with improved chaperone function.

  • 45.
    Dos Reis, Suzana
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pang, Yanhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Vishnu, Neelanjan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Voisset, Cécile
    Galons, Hervé
    Blondel, Marc
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding2011In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 93, no 6, p. 1047-1054Article in journal (Refereed)
    Abstract [en]

    The ribosome, the protein synthesis machinery of the cell, has also been implicated in protein folding. This activity resides within the domain V of the main RNA component of the large subunit of the ribosome. It has been shown that two antiprion drugs 6-aminophenanthridine (GAP) and Guanabenz (GA) bind to the ribosomal RNA and inhibit specifically the protein folding activity of the ribosome. Here, we have characterized with biochemical experiments, the mode of inhibition of these two drugs using ribosomes or ribosomal components active in protein folding (referred to as 'ribosomal folding modulators' or RFMs) from both bacteria Escherichia con and yeast Saccharomyces cerevisiae, and human carbonic anhydrase (HCA) as a sample protein. Our results indicate that 6AP and GA inhibit the protein folding activity of the ribosome by competition with the unfolded protein for binding to the ribosome. As a result, the yield of the refolded protein decreases, but the rate of its refolding remains unaffected. Further, 6AP- and GA mediated inhibition of RFM mediated refolding can be reversed by the addition of RFMs in excess. We also demonstrate with delayed addition of the ribosome and the antiprion drugs that there is a short time-span in the range of seconds within which the ribosome interacts with the unfolded protein. Thus we conclude that the protein folding activity of the ribosome is conserved from bacteria to eukaryotes and most likely the substrate for RFMs is an early refolding state of the target protein.

  • 46. Du, Liping
    et al.
    Villarreal, Seth
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Multigene expression in vivo: Supremacy of large versus small terminators for T7 RNA polymerase2012In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 109, no 4, p. 1043-1050Article in journal (Refereed)
    Abstract [en]

    Designing and building multigene constructs is commonplace in synthetic biology. Yet functional successes at first attempts are rare because the genetic parts are not fully modular. In order to improve the modularity of transcription, we previously showed that transcription termination in vitro by bacteriophage T7 RNA polymerase could be made more efficient by substituting the standard, single, TF large (class I) terminator with adjacent copies of the vesicular stomatitis virus (VSV) small (class II) terminator. However, in vitro termination at the downstream VSV terminator was less efficient than at the upstream VSV terminator, and multigene overexpression in vivo was complicated by unexpectedly inefficient VSV termination within Escherichia coli cells. Here, we address hypotheses raised in that study by showing that VSV or preproparathyroid hormone (PTH) small terminators spaced further apart can work independently (i.e., more efficiently) in vitro, and that VSV and PTH terminations are severely inhibited in vivo. Surprisingly, the difference between class II terminator function in vivo versus in vitro is not due to differences in plasmid supercoiling, as supercoiling had a minimal effect on termination in vitro. We therefore turned to TF terminators for BioBrick synthesis of a pentameric gene construct suitable for overexpression in vivo. This indeed enabled coordinated overexpression and copurification of five His-tagged proteins using the first construct attempted, indicating that this strategy is more modular than other strategies. An application of this multigene overexpression and protein copurification method is demonstrated by supplying five of the six E. coli translation factors required for reconstitution of translation from a single cell line via copurification, greatly simplifying the reconstitution.

  • 47.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Amrein, Beat A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Blaha-Nelson, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Recent advances in QM/MM free energy calculations using reference potentials2015In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1850, no 5, p. 954-965Article, review/survey (Refereed)
    Abstract [en]

    Background: Recent years have seen enormous progress in the development of methods for modeling (bio)molecular systems. This has allowed for the simulation of ever larger and more complex systems. However, as such complexity increases, the requirements needed for these models to be accurate and physically meaningful become more and more difficult to fulfill. The use of simplified models to describe complex biological systems has long been shown to be an effective way to overcome some of the limitations associated with this computational cost in a rational way. Scope of review: Hybrid QM/MM approaches have rapidly become one of the most popular computational tools for studying chemical reactivity in biomolecular systems. However, the high cost involved in performing high-level QM calculations has limited the applicability of these approaches when calculating free energies of chemical processes. In this review, we present some of the advances in using reference potentials and mean field approximations to accelerate high-level QM/MM calculations. We present illustrative applications of these approaches and discuss challenges and future perspectives for the field. Major conclusions: The use of physically-based simplifications has shown to effectively reduce the cost of high-level QM/MM calculations. In particular, lower-level reference potentials enable one to reduce the cost of expensive free energy calculations, thus expanding the scope of problems that can be addressed. General significance: As was already demonstrated 40 years ago, the usage of simplified models still allows one to obtain cutting edge results with substantially reduced computational cost. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

  • 48.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Amrein, Beat Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Modeling catalytic promiscuity in the alkaline phosphatase superfamily2013In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 15, no 27, p. 11160-11177Article in journal (Refereed)
    Abstract [en]

    In recent years, it has become increasingly clear that promiscuity plays a key role in the evolution of new enzyme function. This finding has helped to elucidate fundamental aspects of molecular evolution. While there has been extensive experimental work on enzyme promiscuity, computational modeling of the chemical details of such promiscuity has traditionally fallen behind the advances in experimental studies, not least due to the nearly prohibitive computational cost involved in examining multiple substrates with multiple potential mechanisms and binding modes in atomic detail with a reasonable degree of accuracy. However, recent advances in both computational methodologies and power have allowed us to reach a stage in the field where we can start to overcome this problem, and molecular simulations can now provide accurate and efficient descriptions of complex biological systems with substantially less computational cost. This has led to significant advances in our understanding of enzyme function and evolution in a broader sense. Here, we will discuss currently available computational approaches that can allow us to probe the underlying molecular basis for enzyme specificity and selectivity, discussing the inherent strengths and weaknesses of each approach. As a case study, we will discuss recent computational work on different members of the alkaline phosphatase superfamily (AP) using a range of different approaches, showing the complementary insights they have provided. We have selected this particular superfamily, as it poses a number of significant challenges for theory, ranging from the complexity of the actual reaction mechanisms involved to the reliable modeling of the catalytic metal centers, as well as the very large system sizes. We will demonstrate that, through current advances in methodologies, computational tools can provide significant insight into the molecular basis for catalytic promiscuity, and, therefore, in turn, the mechanisms of protein functional evolution.

  • 49.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Barrozo, Alexandre
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, S. C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Understanding phosphoryl/sulfuryl transfer reactions: from model systems to enzymes2015In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 44, p. S165-S165Article in journal (Other academic)
  • 50.
    Duarte, Fernanda
    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. Univ Oxford, Chem Res Lab, 12 Mansfield Rd, Oxford OX1 3TA, England.;Univ Oxford, Phys & Theoret Chem Lab, S Parks Rd, Oxford OX1 3QZ, England..
    Barrozo, Alexandre
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Williams, Nicholas H.
    Univ Sheffield, Dept Chem, Sheffield S3 7HF, S Yorkshire, England..
    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.
    The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis2016In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 138, no 33, p. 10664-10673Article in journal (Refereed)
    Abstract [en]

    Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group plc on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pK(a) increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pK(a)s studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears.

12345 1 - 50 of 210
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