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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: 2018-12-03Bibliographically approved
    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.

    The full text will be freely available from 2019-01-01 15:28
  • 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: 2018-12-03Bibliographically approved
    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.

    Keywords
    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
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    2018 (English)In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 5, no 3, p. 269-282Article in journal (Refereed) Published
    Abstract [en]

    The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenyl­propane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure–activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.

    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-343750 (URN)10.1107/S2052252518003573 (DOI)000431151300004 ()29755743 (PubMedID)
    Funder
    Swedish Research CouncilEU, FP7, Seventh Framework Programme
    Available from: 2018-03-01 Created: 2018-03-01 Last updated: 2018-12-03Bibliographically approved
    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.

    Keywords
    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