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  • 1.
    Barrozo, Alexandre
    et al.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Liao, Qinghua
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Esguerra, Mauricio
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Marloie, Gael
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Florian, Jan
    Loyola Univ Chicago, Dept Chem & Biochem, Chicago, IL 60660 USA..
    Williams, Nicholas H.
    Univ Sheffield, Dept Chem, Sheffield S3 7HF, S Yorkshire, England..
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Computer simulations of the catalytic mechanism of wild-type and mutant beta-phosphoglucomutase2018Inngår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 16, nr 12, s. 2060-2073Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    beta-Phosphoglucomutase (beta-PGM) has served as an important model system for understanding biological phosphoryl transfer. This enzyme catalyzes the isomerization of beta-glucose-1-phosphate to -glucose-6-phosphate in a two-step process proceeding via a bisphosphate intermediate. The conventionally accepted mechanism is that both steps are concerted processes involving acid-base catalysis from a nearby aspartate (D10) side chain. This argument is supported by the observation that mutation of D10 leaves the enzyme with no detectable activity. However, computational studies have suggested that a substrate-assisted mechanism is viable for many phosphotransferases. Therefore, we carried out empirical valence bond (EVB) simulations to address the plausibility of this mechanistic alternative, including its role in the abolished catalytic activity of the D10S, D10C and D10N point mutants of beta-PGM. In addition, we considered both of these mechanisms when performing EVB calculations of the catalysis of the wild type (WT), H20A, H20Q, T16P, K76A, D170A and E169A/D170A protein variants. Our calculated activation free energies confirm that D10 is likely to serve as the general base/acid for the reaction catalyzed by the WT enzyme and all its variants, in which D10 is not chemically altered. Our calculations also suggest that D10 plays a dual role in structural organization and maintaining electrostatic balance in the active site. The correct positioning of this residue in a catalytically competent conformation is provided by a functionally important conformational change in this enzyme and by the extensive network of H-bonding interactions that appear to be exquisitely preorganized for the transition state stabilization.

    Fulltekst (pdf)
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  • 2.
    Barz, Bogdan
    et al.
    Forschungszentrum Jülich GmbH, Institute of Complex Systems: Structural Biochemistry (ICS-6), Jülich; Heinrich Heine University Düsseldorf, Institute of Theoretical and Computational Chemistry, Düsseldorf.
    Liao, Qinghua
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Forschungszentrum Jülich GmbH, Institute of Complex Systems: Structural Biochemistry (ICS-6), Jülich.
    Strodel, Birgit
    Forschungszentrum Jülich GmbH, Institute of Complex Systems: Structural Biochemistry (ICS-6), Jülich; Heinrich Heine University Düsseldorf, Institute of Theoretical and Computational Chemistry, Düsseldorf.
    Pathways of Amyloid-β Aggregation Depend on Oligomer Shape2018Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, nr 1, s. 319-327Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    One of the main research topics related to Alzheimer’s disease is the aggregation of the amyloid-β peptide, which was shown to follow different pathways for the two major alloforms of the peptide, Aβ40 and the more toxic Aβ42. Experimental studies emphasized that oligomers of specific sizes appear in the early aggregation process in different quantities and might be the key toxic agents for each of the two alloforms. We use transition networks derived from all-atom molecular dynamics simulations to show that the oligomers leading to the type of oligomer distributions observed in experiments originate from compact conformations. Extended oligomers, on the other hand, contribute more to the production of larger aggregates thus driving the aggregation process. We further demonstrate that differences in the aggregation pathways of the two Aβ alloforms occur as early as during the dimer stage. The higher solvent-exposure of hydrophobic residues in Aβ42 oligomers contributes to the different aggregation pathways of both alloforms and also to the increased cytotoxicity of Aβ42.

  • 3.
    Bauer, Paul
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Barrozo, Alexandre
    Department of Chemistry, University of Southern California, SGM 418, 3620 McClintock Ave., Los Angeles, CA 90089-1062, United StatesDepartment of Chemistry, University of Southern California, SGM 418, 3620 McClintock Ave., Los Angeles, CA 90089-1062, United States.
    Purg, Miha
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Amrein, Beat Anton
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Esguerra, Mauricio
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wilson, Philippe Barrie
    Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, UK.
    Major, Dan Thomas
    Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel.
    Åqvist, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Q6: A comprehensive toolkit for empirical valence bond and related free energy calculations2018Inngår i: SoftwareX, ISSN 2352-7110, s. 388-395Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Atomistic simulations have become one of the main approaches to study the chemistry and dynamicsof biomolecular systems in solution. Chemical modelling is a powerful way to understand biochemistry,with a number of different programs available to perform specialized calculations. We present here Q6, anew version of the Q software package, which is a generalized package for empirical valence bond, linearinteraction energy, and other free energy calculations. In addition to general technical improvements, Q6extends the reach of the EVB implementation to fast approximations of quantum effects, extended solventdescriptions and quick estimation of the contributions of individual residues to changes in the activationfree energy of reactions.

    Fulltekst (pdf)
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  • 4.
    Beiroth, Femke
    et al.
    Christiana Albertina Univ Kiel, Otto Diels Inst Organ Chem, Otto Hahn Pl 3-4, D-24118 Kiel, Germany.
    Koudelka, Tomas
    Christiana Albertina Univ Kiel, Inst Expt Med, Systemat Prote & Bioanalyt, Niemannsweg 11, D-24105 Kiel, Germany.
    Overath, Thorsten
    Christiana Albertina Univ Kiel, Inst Expt Med, Systemat Prote & Bioanalyt, Niemannsweg 11, D-24105 Kiel, Germany.
    Knight, Stefan D.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Tholey, Andreas
    Christiana Albertina Univ Kiel, Inst Expt Med, Systemat Prote & Bioanalyt, Niemannsweg 11, D-24105 Kiel, Germany.
    Lindhorst, Thisbe K.
    Christiana Albertina Univ Kiel, Otto Diels Inst Organ Chem, Otto Hahn Pl 3-4, D-24118 Kiel, Germany.
    Diazirine-functionalized mannosides for photoaffinity labeling: trouble with FimH2018Inngår i: Beilstein Journal of Organic Chemistry, ISSN 2195-951X, E-ISSN 1860-5397, Vol. 14, s. 1890-1900Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Photoaffinity labeling is frequently employed for the investigation of ligand-receptor interactions in solution. We have employed an interdisciplinary methodology to achieve facile photolabeling of the lectin FimH, which is a bacterial protein, crucial for adhesion, colonization and infection. Following our earlier work, we have here designed and synthesized diazirine-functionalized mannosides as high-affinity FimH ligands and performed an extensive study on photo-crosslinking of the best ligand (mannoside 3) with a series of model peptides and FimH. Notably, we have employed high-performance mass spectrometry to be able to detect radiation results with the highest possible accuracy. We are concluding from this study that photolabeling of FimH with sugar diazirines has only very limited success and cannot be regarded a facile approach for covalent modification of FimH.

    Fulltekst (pdf)
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  • 5.
    Burke, Jason R.
    et al.
    Salk Inst Biol Studies, Jack H Skirball Ctr Chem Biol & Prote, Howard Hughes Med Inst, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA;Calif State Univ San Bernardino, San Bernardino, CA 92407 USA.
    La Clair, James J.
    Salk Inst Biol Studies, Jack H Skirball Ctr Chem Biol & Prote, Howard Hughes Med Inst, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA.
    Philippe, Ryan N.
    Salk Inst Biol Studies, Jack H Skirball Ctr Chem Biol & Prote, Howard Hughes Med Inst, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA;Manus Biosynth, Cambridge, MA USA.
    Pabis, Anna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Corbella, Marina
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC.
    Jez, Joseph M.
    Salk Inst Biol Studies, Jack H Skirball Ctr Chem Biol & Prote, Howard Hughes Med Inst, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA;Washington Univ, Dept Biol, Howard Hughes Med Inst, Campus Box 1137, St Louis, MO 63130 USA.
    Cortina, George A.
    Univ Virginia, Dept Mol Physiol & Biomed Engn, Charlottesville, VA 22903 USA.
    Kaltenbach, Miriam
    Weizmann Inst Sci, Dept Biomol Sci, IL-76100 Rehovot, Israel.
    Bowman, Marianne E.
    Salk Inst Biol Studies, Jack H Skirball Ctr Chem Biol & Prote, Howard Hughes Med Inst, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA.
    Louie, Gordon V.
    Salk Inst Biol Studies, Jack H Skirball Ctr Chem Biol & Prote, Howard Hughes Med Inst, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA.
    Woods, Katherine B.
    Salk Inst Biol Studies, Jack H Skirball Ctr Chem Biol & Prote, Howard Hughes Med Inst, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA.
    Nelson, Andrew T.
    Univ Texas Austin, Dept Chem, Austin, TX 78712 USA.
    Tawfik, Dan S.
    Weizmann Inst Sci, Dept Biomol Sci, IL-76100 Rehovot, Israel.
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Noel, Joseph P.
    Salk Inst Biol Studies, Jack H Skirball Ctr Chem Biol & Prote, Howard Hughes Med Inst, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA.
    Bifunctional Substrate Activation via an Arginine Residue Drives Catalysis in Chalcone Isomerases2019Inngår i: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 9, nr 9, s. 8388-8396Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Chalcone isomerases are plant enzymes that perform enantioselective oxa-Michael cyclizations of 2'-hydroxychalcones into flavanones. An X-ray crystal structure of an enzyme-product complex combined with molecular dynamics simulations reveal an enzyme mechanism wherein the guanidinium ion of a conserved arginine positions the nucleophilic phenoxide and activates the electrophilic enone for cyclization through Bronsted and Lewis acid interactions. The reaction terminates by asymmetric protonation of the carbanion intermediate syn to the guanidinium. Interestingly, bifunctional guanidine- and urea-based chemical reagents, increasingly used for asymmetric organocatalytic applications, share mechanistic similarities with this natural system. Comparative protein crystal structures and molecular dynamics simulations further demonstrate how two active site water molecules coordinate a hydrogen bond network that enables expanded substrate reactivity for 6'-deoxychalcones in more recently evolved type-2 chalcone isomerases.

  • 6.
    Durall de la Fuente, Claudia
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Molekylär biomimetik.
    Kanchugal Puttaswamy, Sandesh
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Lindblad, Peter
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Molekylär biomimetik.
    Phosphoenolpyruvate carboxylase in Synechococcus PCC 7002: Oligomerization, structure, and characteristics2020Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 10, artikkel-id 3607Artikkel i tidsskrift (Fagfellevurdert)
    Fulltekst (pdf)
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  • 7.
    Elison Kalman, Grim
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Purification, functional characterization and crystallization of the PerR peroxide sensor from Saccharopolyspora erythraea2019Independent thesis Advanced level (degree of Master (Two Years)), 20 poäng / 30 hpOppgave
    Abstract [en]

    This report summarizes the work on the cloning, expression, and purification of PerR, a metal sensing regulator from Saccharopolyspora erythraea and the subsequent characterization using small angle X-ray scattering and other biochemical methods. The report aims to provide an insight into prokaryotic metal homeostasis, provide a better understanding of how PerR works and provide valuable information for the continued work on the crystallization of PerR.

    Fulltekst (pdf)
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  • 8.
    Ge, Xueliang
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
    Oliveira, Ana
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Hjort, Karin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Bergfors, Terese
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Gutiérrez-de-Terán, Hugo
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Andersson, Dan I
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Sanyal, Suparna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylärbiologi.
    Åqvist, Johan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Inhibition of translation termination by small molecules targeting ribosomal release factors2019Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikkel-id 15424Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

    Fulltekst (pdf)
    fulltext
  • 9.
    Griese, Julia J.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden.
    Branca, Rui M. M.
    Karolinska Inst, Sci Life Lab, Dept Oncol Pathol, Canc Prote Mass Spectrometry, Box 1031, S-17121 Solna, Sweden.
    Srinivas, Vivek
    Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden.
    Hogbom, Martin
    Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden.
    Ether cross-link formation in the R2-like ligand-binding oxidase2018Inngår i: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 23, nr 6, s. 879-886Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of Mn-II and Fe-II in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine-valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.

    Fulltekst (pdf)
    fulltext
  • 10.
    Griese, Julia J.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
    Högbom, Martin
    Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
    Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion: Q-XAD2019Inngår i: Acta Crystallographica Section D: Structural Biology, ISSN 2059-7983, Vol. 75, nr 8, s. 764-771Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a hetero­dinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of high-quality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided.

    Fulltekst (pdf)
    fulltext
  • 11.
    Griese, Julia J.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden.
    Kositzki, Ramona
    Free Univ Berlin, Inst Expt Phys, D-14195 Berlin, Germany.
    Haumann, Michael
    Free Univ Berlin, Inst Expt Phys, D-14195 Berlin, Germany.
    Hogbom, Martin
    Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden.
    Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine-valine ether cross-link formation in the R2-like ligand-binding oxidase2019Inngår i: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 24, nr 2, s. 211-221Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O-2) activation, catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O-2, but prefers manganese in the presence of O-2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O-2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O-2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.

    Fulltekst (pdf)
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  • 12.
    Grāve, Kristīne
    et al.
    Stockholm University.
    Lambert, Wietske
    PRA Health Sciences, Assen, The Netherlands.
    Berggren, Gustav
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Molekylär biomimetik.
    Griese, Julia J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Department of Biochemistry and Biophysics, Stockholm University.
    Bennett, Matthew D.
    Stockholm University.
    Logan, Derek T.
    Lund University.
    Högbom, Martin
    Stockholm University.
    Redox-induced structural changes in the di-iron and di-manganese forms of Bacillus anthracis ribonucleotide reductase subunit NrdF suggest a mechanism for gating of radical access2019Inngår i: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 24, nr 6, s. 849-861Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (MnII/MnII, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (FeII/FeII form, 1.32 Å), or prepared aerobically in the photo-reduced FeII/FeII form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.

    Fulltekst (pdf)
    fulltext
  • 13. Guo, Xiaohu
    et al.
    Söderholm, Annika
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Kanchugal Puttaswamy, Sandesh
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Isaksen, Geir
    Hylleraas Centre for Quantum Molecular Sciences, Department of Chemistry, UiT - The Arctic University of Norway, N9037, Tromsø, Norway.
    Warsi, Omar M.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eckhard, Ulrich
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Trigüis, Silvia
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Gogoll, A
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi.
    Jerlstrom-Hultqvist, Joel
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Åqvist, Johan
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Andersson, Dan I
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Structure of a phage-encoded SAMase enzyme provides insights in substrate binding and mechanismManuskript (preprint) (Annet vitenskapelig)
  • 14.
    Harish, Ajith
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Kurland, Charles
    Lund Univ, Dept Biol, Lund, Sweden.
    Reply to Caetano-Anolles et al. comment on "Empirical genome evolution models root the tree of life"2018Inngår i: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 149, s. 137-138Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    We recently analyzed the robustness of competing evolution models developed to identify the root of the Tree of Life: 1) An empirical Sankoff parsimony (ESP) model (Harish and Kurland, 2017), which is a nonstationary and directional evolution model; and 2) An a priori ancestor (APA) model (Kim and Caetano-Anolles, 2011) that is a stationary and reversible evolution model. Both Bayesian model selection tests as well as maximum parsimony analyses demonstrate that the ESP model is, overwhelmingly, the better model. Moreover, we showed that the APA model is not only sensitive to artifacts, but also that the underlying assumptions are neither empirically grounded nor biologically realistic.

  • 15.
    Harish, Ajith
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Kurland, Charles G.
    Lund Univ, Sect Microbial Ecol, Dept Biol, Lund, Sweden..
    Mitochondria are not captive bacteria2017Inngår i: Journal of Theoretical Biology, ISSN 0022-5193, E-ISSN 1095-8541, Vol. 434, s. 88-98Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Lynn Sagan's conjecture (1967) that three of the fundamental organelles observed in eukaryote cells, specifically mitochondria, plastids and flagella were once free-living primitive (prokaryotic) cells was accepted after considerable opposition. Even though the idea was swiftly refuted for the specific case of origins of flagella in eukaryotes, the symbiosis model in general was accepted for decades as a realistic hypothesis to describe the endosymbiotic origins of eukaryotes. However, a systematic analysis of the origins of the mitochondrial proteome based on empirical genome evolution models now indicates that 97% of modern mitochondrial protein domains as well their homologues in bacteria and archaea were present in the universal common ancestor (UCA) of the modern tree of life (ToL). These protein domains are universal modular building blocks of modern genes and genomes, each of which is identified by a unique tertiary structure and a specific biochemical function as well as a characteristic sequence profile. Further, phylogeny reconstructed from genome-scale evolution models reveals that Eukaryotes and Akaryotes (archaea and bacteria) descend independently from UCA. That is to say, Eukaryotes and Akaryotes are both primordial lineages that evolved in parallel. Finally, there is no indication of massive inter-lineage exchange of coding sequences during the descent of the two lineages. Accordingly, we suggest that the evolution of the mitochondrial proteome was autogenic (endogenic) and not endosymbiotic (exogenic).

  • 16.
    Janfalk Carlsson, Åsa
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Bauer, Paul
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Dobritzsch, Doreen
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Kamerlin, Shina C Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme2018Inngår i: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 5, nr 3, s. 269-282Artikkel i tidsskrift (Fagfellevurdert)
    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.

    Fulltekst (pdf)
    fulltext
  • 17.
    Jerlström-Hultqvist, Joel
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
    Warsi, Omar
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Söderholm, Annika
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Knopp, Michael
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eckhard, Ulrich
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Vorontsov, Egor
    Proteomics Core Facility at Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Andersson, Dan I
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    A bacteriophage enzyme induces bacterial metabolic perturbation that confers a novel promiscuous function2018Inngår i: Nature Ecology & Evolution, E-ISSN 2397-334X, Vol. 2, nr 8, s. 1321-1330Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    One key concept in the evolution of new functions is the ability of enzymes to perform promiscuous side-reactions that serve as a source of novelty that may become beneficial under certain conditions. Here, we identify a mechanism where a bacteriophage-encoded enzyme introduces novelty by inducing expression of a promiscuous bacterial enzyme. By screening for bacteriophage DNA that rescued an auxotrophic Escherichia coli mutant carrying a deletion of the ilvA gene, we show that bacteriophage-encoded S-adenosylmethionine (SAM) hydrolases reduce SAM levels. Through this perturbation of bacterial metabolism, expression of the promiscuous bacterial enzyme MetB is increased, which in turn complements the absence of IlvA. These results demonstrate how foreign DNA can increase the metabolic capacity of bacteria, not only by transfer of bona fide new genes, but also by bringing cryptic bacterial functions to light via perturbations of cellular physiology.

  • 18.
    Jiang, Wangshu
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Of spiders, bugs, and men: Structural and functional studies of proteins involved in assembly2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Protein assembly enables complex machineries while being economical with genetic information. However, protein assembly also constitutes a potential threat to the host, and needs to be carefully regulated.

    Sulfate is a common source of sulfur for cysteine synthesis in bacteria. A putative sulfate permease CysZ from Escherichia coli appears much larger than its apparent molecular mass when analyzed by chromatography and native gel. Clearly CysZ undergoes homo-oligomerization. Using isothermal titration calorimetry, we confirmed that CysZ binds to its putative substrate sulfate, and also sulfite with higher affinity. CysZ-mediated sulfate transport—in both E. coli whole cells and proteoliposomes—was inhibited in the presence of sulfite, indicating a feedback inhibition mechanism.

    Proteus mirabilis is a Gram-negative bacterium causing urinary tract infections. Its simultaneous expression of multiple fimbriae enables colonization and biofilm formation. Fimbriae are surface appendages assembled from protein subunits, with distal adhesins specifically recognizing host-cell receptors. We present the first three structures of P. mirabilis fimbrial adhesins. While UcaD and AtfE adopt the canonical immunoglobulin-like fold, MrpH has a previously unknown fold. The coordination of Zn or Cu ion by three conserved histidine residues in MrpH is required for MrpH-dependent biofilm formation.

    Spider silk is an assembly of large proteins called spidroins. The N-terminal domain (NT) of spidroins senses the pH decrease along the silk spinning gland, and transits from monomer to dimer. A locked NT dimer interlinks spidroin molecules into polymers. We identified a new asymmetric dimer form of NT by x-ray crystallography. With additional evidence from small angle x-ray scattering (SAXS), we propose the asymmetric dimer as a common intermediate of NT in silk formation.

    Alzheimer’s disease is a life-threatening dementia, where aggregation-prone Aβ peptides self-assemble into amyloid fibrils. Bri2 BRICHOS is a molecular chaperone that efficiently delays Aβ fibrillation, and protects the region of its pro-protein with high β-propensity from aggregation. Combining SAXS and microscale thermophoresis data, we confirmed binding between Bri2 BRICHOS and its native client peptide. Using site-directed mutagenesis, we showed that three conserved tyrosine residues in Bri2 BRICHOS are important for its anti-Aβ fibrillation activity.

    Delarbeid
    1. The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite
    Åpne denne publikasjonen i ny fane eller vindu >>The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite
    Vise andre…
    2014 (engelsk)Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, nr 7, s. 1809-1816Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    The Escherichia coli inner membrane protein CysZ mediates the sulfate uptake subsequently utilized for the synthesis of sulfur-containing compounds in cells. Here we report the purification and functional characterization of CysZ. Using Isothermal Titration Calorimetry, we have observed interactions between CysZ and its putative substrate sulfate. Additional sulfur-containing compounds from the cysteine synthesis pathway have also been analyzed for their abilities to interact with CysZ. Our results suggest that CysZ is dedicated to a specific pathway that assimilates sulfate for the synthesis of cysteine. Sulfate uptake via CysZ into E. coil whole cells and proteoliposome offers direct evidence of CysZ being able to mediate sulfate uptake. In addition, the cysteine synthesis pathway intermediate sulfite can interact directly with CysZ with higher affinity than sulfate. The sulfate transport activity is inhibited in the presence of sulfite, suggesting the existence of a feedback inhibition mechanism in which sulfite regulates sulfate uptake by CysZ. Sulfate uptake assays performed at different extracellular pH and in the presence of a proton uncoupler indicate that this uptake is driven by the proton gradient. (C) 2014 Elsevier B.V. All rights reserved.

    Emneord
    CysZ, Sulfate, Transport, Membrane protein, Inhibition
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-227987 (URN)10.1016/j.bbamem.2014.03.003 (DOI)000336695300014 ()
    Tilgjengelig fra: 2014-07-04 Laget: 2014-07-02 Sist oppdatert: 2018-11-23bibliografisk kontrollert
    2. Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis
    Åpne denne publikasjonen i ny fane eller vindu >>Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis
    2018 (engelsk)Inngår i: Acta crystallographica. Section D, Structural biology, ISSN 2059-7983, Vol. 74, nr Pt 11, s. 1053-1062Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    The important uropathogen Proteus mirabilis encodes a record number of chaperone/usher-pathway adhesive fimbriae. Such fimbriae, which are used for adhesion to cell surfaces/tissues and for biofilm formation, are typically important virulence factors in bacterial pathogenesis. Here, the structures of the receptor-binding domains of the tip-located two-domain adhesins UcaD (1.5 Å resolution) and AtfE (1.58 Å resolution) from two P. mirabilis fimbriae (UCA/NAF and ATF) are presented. The structures of UcaD and AtfE are both similar to the F17G type of tip-located fimbrial receptor-binding domains, and the structures are very similar despite having only limited sequence similarity. These structures represent an important step towards a molecular-level understanding of P. mirabilis fimbrial adhesins and their roles in the complex pathogenesis of urinary-tract infections.

    Emneord
    Proteus mirabilis, adhesins, fimbriae, urinary-tract infection
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-366693 (URN)10.1107/S2059798318012391 (DOI)000449044300003 ()30387764 (PubMedID)
    Forskningsfinansiär
    Swedish Research Council
    Tilgjengelig fra: 2018-11-22 Laget: 2018-11-22 Sist oppdatert: 2019-01-07bibliografisk kontrollert
    3. Structural basis for MrpH-dependent Proteus mirabilis biofilm formation
    Åpne denne publikasjonen i ny fane eller vindu >>Structural basis for MrpH-dependent Proteus mirabilis biofilm formation
    Vise andre…
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Proteus mirabilis is a Gram-negative uropathogen and the major causative agent in catheter-associated  (CAUTI) and complicated UTIs. Mannose resistant Proteus-like fimbriae (MR/P) are crucially important for P. mirabilis infectivity and are required for biofilm formation and auto-aggregation, as well as for bladder and kidney colonisation. Here, the X-ray structure of the MR/P tip-located MrpH adhesin is reported. The structure has an unusual fold not previously observed, and contains a transition metal centre with Cu2+ or Zn2+ ligated by three conserved histidine residues and a ligand. Using metal complementation biofilm assays and site directed mutagenesis of the three histidines we show that an intact metal binding site occupied by zinc or copper is essential for MR/P-mediated biofilm formation. The studies presented here provide important clues as to the mechanism of MR/P-mediated biofilm formation and will serve as a starting point for identifying the physiological MR/P receptor(s).

    Emneord
    fimbriae; adhesins; biofilm; urinary tract infection; Proteus mirabilis
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-366696 (URN)
    Tilgjengelig fra: 2018-11-22 Laget: 2018-11-22 Sist oppdatert: 2018-11-23
    4. Conversion of spidroin dope to spider silk involves an asymmetric dimer intermediate
    Åpne denne publikasjonen i ny fane eller vindu >>Conversion of spidroin dope to spider silk involves an asymmetric dimer intermediate
    Vise andre…
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-366699 (URN)
    Tilgjengelig fra: 2018-11-22 Laget: 2018-11-22 Sist oppdatert: 2018-11-23
    5. Bri2 BRICHOS domain binds Bri23 and depends on conserved face A tyrosine residues for anti-amyloid activity
    Åpne denne publikasjonen i ny fane eller vindu >>Bri2 BRICHOS domain binds Bri23 and depends on conserved face A tyrosine residues for anti-amyloid activity
    Vise andre…
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    Emneord
    Bri2, BRICHOS, amyloid, Alzheimer's disease, molecular chaperone
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-366698 (URN)
    Tilgjengelig fra: 2018-11-22 Laget: 2018-11-22 Sist oppdatert: 2018-11-23
    Fulltekst (pdf)
    fulltext
    Download (jpg)
    presentationsbild
  • 19.
    Jiang, Wangshu
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Askarieh, Glareh
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Shkumatov, Alexander
    Structural Biology Brussels, Belgium.
    Hedhammar, My
    KTH royal institute of technology.
    Knight, Stefan D
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Conversion of spidroin dope to spider silk involves an asymmetric dimer intermediateManuskript (preprint) (Annet vitenskapelig)
  • 20.
    Jiang, Wangshu
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Askarieh, Glareh
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Shkumatov, Alexander
    Vrije Univ Brussel, Struct Biol Brussels, B-1050 Brussels, Belgium;VIB VUB Ctr Struct Biol, B-1050 Brussels, Belgium.
    Hedhammar, My
    KTH Royal Inst Technol, Div Prot Technol, Alballova Univ Ctr, Roslagstullsbacken 21, S-10691 Stockholm, Sweden.
    Knight, Stefan D.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Structure of the N-terminal domain of Euprosthenops australis dragline silk suggests that conversion of spidroin dope to spider silk involves a conserved asymmetric dimer intermediate2019Inngår i: ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, ISSN 2059-7983, Vol. 75, s. 618-627Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values. Conversion from the monomer to the dimer requires the protonation of three conserved glutamic acid residues, resulting in a low-pH 'locked' dimer stabilized by symmetric electrostatic interactions at the poles of the dimer. The detailed molecular events during this transition are still unresolved. Here, a 2.1 angstrom resolution crystal structure of an NT T61A mutant in an alternative, asymmetric, dimer form in which the electrostatic interactions at one of the poles are dramatically different from those in symmetrical dimers is presented. A similar asymmetric dimer structure from dragline silk of Nephila clavipes has previously been described. It is suggested that asymmetric dimers represent a conserved intermediate state in spider silk formation, and a revised 'lock-and-trigger' mechanism for spider silk formation is presented.

  • 21.
    Jiang, Wangshu
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Chen, Gefei
    Karolinska Institutet.
    Achterhold, Alina
    TU Bergakademie Freiberg.
    Johansson, Jan
    Department of Neurobiology, Care sciences and Society, Center for Alzheimer Research, Karolinska Institutet.
    Knight, Stefan D
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Bri2 BRICHOS domain binds Bri23 and depends on conserved face A tyrosine residues for anti-amyloid activityManuskript (preprint) (Annet vitenskapelig)
  • 22.
    Jiang, Wangshu
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Ubhayasekera, Wimal
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Breed, Michael C
    University of Michigan Medical School.
    Serr, Nina
    University of Michigan Medical School.
    Pearson, Melanie M
    University of Michigan Medical School.
    Knight, Stefan D
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Structural basis for MrpH-dependent Proteus mirabilis biofilm formationManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Proteus mirabilis is a Gram-negative uropathogen and the major causative agent in catheter-associated  (CAUTI) and complicated UTIs. Mannose resistant Proteus-like fimbriae (MR/P) are crucially important for P. mirabilis infectivity and are required for biofilm formation and auto-aggregation, as well as for bladder and kidney colonisation. Here, the X-ray structure of the MR/P tip-located MrpH adhesin is reported. The structure has an unusual fold not previously observed, and contains a transition metal centre with Cu2+ or Zn2+ ligated by three conserved histidine residues and a ligand. Using metal complementation biofilm assays and site directed mutagenesis of the three histidines we show that an intact metal binding site occupied by zinc or copper is essential for MR/P-mediated biofilm formation. The studies presented here provide important clues as to the mechanism of MR/P-mediated biofilm formation and will serve as a starting point for identifying the physiological MR/P receptor(s).

  • 23.
    Jiang, Wangshu
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Ubhayasekera, Wimal
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Pearson, Melanie M.
    Univ Michigan, Sch Med, Dept Microbiol & Immunol, Ann Arbor, MI USA.
    Knight, Stefan D.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis2018Inngår i: Acta crystallographica. Section D, Structural biology, ISSN 2059-7983, Vol. 74, nr Pt 11, s. 1053-1062Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The important uropathogen Proteus mirabilis encodes a record number of chaperone/usher-pathway adhesive fimbriae. Such fimbriae, which are used for adhesion to cell surfaces/tissues and for biofilm formation, are typically important virulence factors in bacterial pathogenesis. Here, the structures of the receptor-binding domains of the tip-located two-domain adhesins UcaD (1.5 Å resolution) and AtfE (1.58 Å resolution) from two P. mirabilis fimbriae (UCA/NAF and ATF) are presented. The structures of UcaD and AtfE are both similar to the F17G type of tip-located fimbrial receptor-binding domains, and the structures are very similar despite having only limited sequence similarity. These structures represent an important step towards a molecular-level understanding of P. mirabilis fimbrial adhesins and their roles in the complex pathogenesis of urinary-tract infections.

  • 24.
    Kaltenbach, Miriam
    et al.
    Weizmann Inst Sci, Dept Biol Chem, Rehovot, Israel.
    Burke, Jason R.
    Salk Inst Biol Studies, Howard Hughes Med Inst, Jack H Skirball Ctr Chem Biol & Prote, La Jolla, CA 92037 USA.
    Dindo, Mirco
    Weizmann Inst Sci, Dept Biol Chem, Rehovot, Israel;Univ Verona, Biol Chem Sect, Dept Neurosci Biomed & Movement Sci, Verona, Italy.
    Pabis, Anna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Steffen-Munsberg, Fabian
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Rabin, Avigayel
    Weizmann Inst Sci, Dept Biol Chem, Rehovot, Israel;Hebrew Univ Jerusalem, Alexander Silberman Inst Life Sci, Dept Biol Chem, Edmond J Safra Campus, Jerusalem, Israel.
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Noel, Joseph P.
    Salk Inst Biol Studies, Howard Hughes Med Inst, Jack H Skirball Ctr Chem Biol & Prote, La Jolla, CA 92037 USA.
    Tawfik, Dan S.
    Weizmann Inst Sci, Dept Biol Chem, Rehovot, Israel.
    Evolution of chalcone isomerase from a noncatalytic ancestor2018Inngår i: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, nr 6, s. 548-555Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The emergence of catalysis in a noncatalytic protein scaffold is a rare, unexplored event. Chalcone isomerase (CHI), a key enzyme in plant flavonoid biosynthesis, is presumed to have evolved from a nonenzymatic ancestor related to the widely distributed fatty-acid binding proteins (FAPs) and a plant protein family with no isomerase activity (CHILs). Ancestral inference supported the evolution of CHI from a protein lacking isomerase activity. Further, we identified four alternative founder mutations, i.e., mutations that individually instated activity, including a mutation that is not phylogenetically traceable. Despite strong epistasis in other cases of protein evolution, CHI's laboratory reconstructed mutational trajectory shows weak epistasis. Thus, enantioselective CHI activity could readily emerge despite a catalytically inactive starting point. Accordingly, X-ray crystallography, NMR, and molecular dynamics simulations reveal reshaping of the active site toward a productive substratebinding mode and repositioning of the catalytic arginine that was inherited from the ancestral fatty-acid binding proteins.

    Fulltekst (pdf)
    fulltext
  • 25.
    Kanchugal P, Sandesh
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Antibiotic Resistance and the Cellular Currency S-adenosyl-methionine: Modification of aminoglycosides and nucleic acids2020Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Streptomycin and spectinomycin are antibiotics that bind to ribosomes and inhibit protein synthesis. Common resistance mechanisms involve enzymatic modification of the two drugs by aminoglycoside nucleotidyltransferases (ANTs). The first part of this thesis covers the structural mechanism of two ANT enzymes. The first is the dual-specificity AadA, belonging to the ANT(3")(9) family, which modifies the 3" position of streptomycin and position 9 of spectinomycin. The second is ANT(9) that only modifies spectinomycin at position 9.

    We solved crystal structures of both enzymes, AadA in complex with ATP and streptomycin and ANT(9) with ATP and spectinomycin. The two enzymes show overall structural similarity and both consist of an N-terminal nucleotidyltransferase domain and a C-terminal helical domain. The binding of ATP between the two domains induces a conformational change that allows the drug to bind. The modified hydroxyl groups of both drugs align at similar positions in the active site, even though the drugs are chemically distinct. Comparison of the ANT(9) and AadA structures shows that spectinomycin specificity is explained by the straight α5 helix followed by a short loop in ANT(9) that would clash with larger drug streptomycin. These findings allowed us to explain the substrate recognition of these enzymes and propose a catalytic mechanism.

    In the second and third parts of this thesis, I studied two enzymes that use S-adenosyl-methionine (SAM), RlmF in site-specific methylation of ribosomal RNA (rRNA) and Svi3-3 in SAM degradation. SAM is an essential molecule for normal cellular function in all-living cells and termed as a ‘cellular currency’. Knowledge is lacking about the substrate recognition of rRNA methyltransferases and the role of the modifications that they add during ribosome assembly. Here, we identify the residues of RlmF that are critical for binding of the cofactor SAM and the lithium chloride core particle substrate that mimics a 50S ribosome assembly intermediate.In the third part, I present structural and ligand-binding studies of a newly discovered SAM degrading enzyme Svi3-3 from bacteriophage.

    Delarbeid
    1. Structural mechanism of AadA, a dual specificity aminoglycoside adenylyltransferase from Salmonella enterica
    Åpne denne publikasjonen i ny fane eller vindu >>Structural mechanism of AadA, a dual specificity aminoglycoside adenylyltransferase from Salmonella enterica
    Vise andre…
    2018 (engelsk)Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, s. 11481-11490Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3’’)(9) adenylyl transferase that O-adenylates position 3” of streptomycin and position 9 of spectinomycin. We previously reported the apo AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of wildtype AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site, and, in agreement with mutational data, critically interacts with fewer residues. Using structure-guided sequence analyses of ANT(3”)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the non-adenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.

    HSV kategori
    Forskningsprogram
    Biologi med inriktning mot strukturbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-353761 (URN)10.1074/jbc.RA118.003989 (DOI)000439449700018 ()29871922 (PubMedID)
    Forskningsfinansiär
    Knut and Alice Wallenberg FoundationSwedish Research Council, 2017-03827Swedish Research Council, 2013-05930EU, FP7, Seventh Framework Programme, 283570
    Tilgjengelig fra: 2018-06-15 Laget: 2018-06-15 Sist oppdatert: 2020-04-14bibliografisk kontrollert
    2. Structural Recognition of Spectinomycin by ResistanceEnzyme ANT(9) from Enterococcus faecalis
    Åpne denne publikasjonen i ny fane eller vindu >>Structural Recognition of Spectinomycin by ResistanceEnzyme ANT(9) from Enterococcus faecalis
    (engelsk)Inngår i: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596Artikkel i tidsskrift (Fagfellevurdert) In press
    Abstract [en]

    Spectinomycin is a ribosome-binding antibiotic that blocks the translocation

    step of translation. A prevalent resistance mechanism is modification of the drug by

    aminoglycoside nucleotidyl transferase (ANT) enzymes of the spectinomycin-specific

    ANT(9) family or by enzymes of the dual-specificity ANT(3)(9) family, which also acts on

    streptomycin. We previously reported the structural mechanism of streptomycin modification

    by the ANT(3)(9) AadA from Salmonella enterica. ANT(9) from Enterococcus

    faecalis adenylates the 9-hydroxyl of spectinomycin. Here, we present the first structures

    of spectinomycin bound to an ANT enzyme. Structures were solved for ANT(9)

    in apo- form, in complex with ATP, spectinomycin, and magnesium, or in complex

    with only spectinomycin. ANT(9) shows an overall structure similar to that of AadA,

    with an N-terminal nucleotidyltransferase domain and a C-terminal -helical domain.

    Spectinomycin binds close to the entrance of the interdomain cleft, while ATP is

    buried at the bottom. Upon drug binding, the C-terminal domain rotates 14 degrees

    to close the cleft, allowing contacts of both domains with the drug. Comparison

    with AadA shows that spectinomycin specificity is explained by a straight 5 helix

    and a shorter 5-6 loop, which would clash with the larger streptomycin substrate.

    In the active site, we observed two magnesium ions, one of them in a previously

    unobserved position that may activate the 9-hydroxyl for deprotonation by the catalytic

    base Glu-86. The observed binding mode for spectinomycin suggests that spectinamides

    and aminomethyl spectinomycins, recent spectinomycin analogues with

    expansions in position 4 of the C ring, are also subjected to modification by ANT(9)

    and ANT(3)(9) enzymes.

    Emneord
    Antibiotic resistance
    HSV kategori
    Forskningsprogram
    Biologi med inriktning mot strukturbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-408616 (URN)10.1128/AAC.00371-20 (DOI)
    Tilgjengelig fra: 2020-04-09 Laget: 2020-04-09 Sist oppdatert: 2020-04-14
    3. Substrate recognition by 23S RNA methyltransferase RlmF
    Åpne denne publikasjonen i ny fane eller vindu >>Substrate recognition by 23S RNA methyltransferase RlmF
    Vise andre…
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    HSV kategori
    Forskningsprogram
    Biologi med inriktning mot strukturbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-408617 (URN)
    Tilgjengelig fra: 2020-04-10 Laget: 2020-04-10 Sist oppdatert: 2020-04-14
    4. Structure of a phage-encoded SAMase enzyme provides insights in substrate binding and mechanism
    Åpne denne publikasjonen i ny fane eller vindu >>Structure of a phage-encoded SAMase enzyme provides insights in substrate binding and mechanism
    Vise andre…
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-408808 (URN)
    Tilgjengelig fra: 2020-04-14 Laget: 2020-04-14 Sist oppdatert: 2020-04-14
    Fulltekst (pdf)
    fulltext
    Download (jpg)
    presentationsbild
  • 26.
    Kanchugal P, Sandesh
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala University.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Structural Recognition of Spectinomycin by ResistanceEnzyme ANT(9) from Enterococcus faecalisInngår i: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Spectinomycin is a ribosome-binding antibiotic that blocks the translocation

    step of translation. A prevalent resistance mechanism is modification of the drug by

    aminoglycoside nucleotidyl transferase (ANT) enzymes of the spectinomycin-specific

    ANT(9) family or by enzymes of the dual-specificity ANT(3)(9) family, which also acts on

    streptomycin. We previously reported the structural mechanism of streptomycin modification

    by the ANT(3)(9) AadA from Salmonella enterica. ANT(9) from Enterococcus

    faecalis adenylates the 9-hydroxyl of spectinomycin. Here, we present the first structures

    of spectinomycin bound to an ANT enzyme. Structures were solved for ANT(9)

    in apo- form, in complex with ATP, spectinomycin, and magnesium, or in complex

    with only spectinomycin. ANT(9) shows an overall structure similar to that of AadA,

    with an N-terminal nucleotidyltransferase domain and a C-terminal -helical domain.

    Spectinomycin binds close to the entrance of the interdomain cleft, while ATP is

    buried at the bottom. Upon drug binding, the C-terminal domain rotates 14 degrees

    to close the cleft, allowing contacts of both domains with the drug. Comparison

    with AadA shows that spectinomycin specificity is explained by a straight 5 helix

    and a shorter 5-6 loop, which would clash with the larger streptomycin substrate.

    In the active site, we observed two magnesium ions, one of them in a previously

    unobserved position that may activate the 9-hydroxyl for deprotonation by the catalytic

    base Glu-86. The observed binding mode for spectinomycin suggests that spectinamides

    and aminomethyl spectinomycins, recent spectinomycin analogues with

    expansions in position 4 of the C ring, are also subjected to modification by ANT(9)

    and ANT(3)(9) enzymes.

  • 27.
    Kanchugal Puttaswamy, Sandesh
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Larsson, Daniel S D
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution. Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Punekar, Avinash S.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Backbro, K
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturell molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Substrate recognition by 23S RNA methyltransferase RlmFManuskript (preprint) (Annet vitenskapelig)
  • 28.
    Kisgeropoulos, Effie C.
    et al.
    Ohio State Univ, Ohio State Biochem Program, Columbus, OH 43210 USA..
    Griese, Julia J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden.
    Smith, Zachary R.
    Ohio State Univ, Dept Chem & Biochem, Columbus, OH 43210 USA..
    Branca, Rui M. M.
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, SE-17121 Solna, Sweden..
    Schneider, Camille R.
    Ohio State Univ, Ohio State Biochem Program, Columbus, OH 43210 USA..
    Hogbom, Martin
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden..
    Shafaat, Hannah S.
    Ohio State Univ, Dept Chem & Biochem, Columbus, OH 43210 USA.;Ohio State Univ, Ohio State Biochem Program, Columbus, OH 43210 USA..
    Key Structural Motifs Balance Metal Binding and Oxidative Reactivity in a Heterobimetallic Mn/Fe Protein2020Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 142, nr 11, s. 5338-5354Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Heterobimetallic Mn/Fe proteins represent a new cofactor paradigm in bioinorganic chemistry and pose countless outstanding questions. The assembly of the active site defies common chemical convention by contradicting the Irving-Williams series, while the scope of reactivity remains unexplored. In this work, the assembly and C-H bond activation process in the Mn/Fe R2-like ligand-binding oxidase (R2lox) protein is investigated using a suite of biophysical techniques, including time-resolved optical spectroscopy, global kinetic modeling, X-ray crystallography, electron paramagnetic resonance spectroscopy, protein electrochemistry, and mass spectrometry. Selective metal binding is found to be under thermodynamic control, with the binding sites within the apoprotein exhibiting greater Mn-II affinity than Fe-II affinity. The comprehensive analysis of structure and reactivity of wild-type R2lox and targeted primary and secondary sphere mutants indicate that the efficiency of C-H bond activation directly correlates with the Mn/Fe cofactor reduction potentials and is inversely related to divalent metal binding affinity. These findings suggest the R2lox active site is precisely tuned for achieving both selective heterobimetallic binding and high levels of reactivity and offer a mechanism to examine the means by which proteins achieve appropriate metal incorporation.

  • 29.
    Koster, Anna K.
    et al.
    Stanford Univ, Dept Chem, Stanford, CA 94305 USA;Stanford Univ, Dept Mol & Cellular Physiol, Sch Med, Stanford, CA 94305 USA.
    Wood, Chase A. P.
    Stanford Univ, Dept Mol & Cellular Physiol, Sch Med, Stanford, CA 94305 USA.
    Thomas-Tran, Rhiannon
    Stanford Univ, Dept Chem, Stanford, CA 94305 USA.
    Chavan, Tanmay S.
    Stanford Univ, Dept Mol & Cellular Physiol, Sch Med, Stanford, CA 94305 USA.
    Almqvist, Jonas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Choi, Kee-Hyun
    Korea Inst Sci & Technol, Mat & Life Sci Res Div, Seoul 02792, South Korea.
    Du Bois, J.
    Stanford Univ, Dept Chem, Stanford, CA 94305 USA.
    Maduke, Merritt
    Stanford Univ, Dept Mol & Cellular Physiol, Sch Med, Stanford, CA 94305 USA.
    A selective class of inhibitors for the CLC-Ka chloride ion channel2018Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, nr 21, s. E4900-E4909Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    CLC proteins are a ubiquitously expressed family of chloride-selective ion channels and transporters. A dearth of pharmacological tools for modulating CLC gating and ion conduction limits investigations aimed at understanding CLC structure/function and physiology. Herein, we describe the design, synthesis, and evaluation of a collection of N-arylated benzimidazole derivatives (BIMs), one of which (BIM1) shows unparalleled (>20-fold) selectivity for CLC-Ka over CLC-Kb, the two most closely related human CLC homologs. Computational docking to a CLC-Ka homology model has identified a BIM1 binding site on the extracellular face of the protein near the chloride permeation pathway in a region previously identified as a binding site for other less selective inhibitors. Results from site-directed mutagenesis experiments are consistent with predictions of this docking model. The residue at position 68 is 1 of only similar to 20 extracellular residues that differ between CLC-Ka and CLC-Kb. Mutation of this residue in CLC-Ka and CLC-Kb (N68D and D68N, respectively) reverses the preference of BIM1 for CLC-Ka over CLC-Kb, thus showing the critical role of residue 68 in establishing BIM1 selectivity. Molecular docking studies together with results from structure-activity relationship studies with 19 BIM derivatives give insight into the increased selectivity of BIM1 compared with other inhibitors and identify strategies for further developing this class of compounds.

  • 30.
    Kulkarni, Yashraj
    et al.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Amyes, Tina L.
    SUNY Buffalo, Dept Chem, Buffalo, NY 14260 USA.
    Richard, John P.
    SUNY Buffalo, Dept Chem, Buffalo, NY 14260 USA.
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Uncovering the Role of Key Active-Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalyzed by Wild-Type and Variants of Triosephosphate Isomerase2019Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 141, nr 40, s. 16139-16150Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We report results of detailed empirical valence bond simulations that model the effect of several amino acid substitutions on the thermodynamic (ΔG°) and kinetic activation (ΔG) barriers to deprotonation of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) bound to wild-type triosephosphate isomerase (TIM), as well as to the K12G, E97A, E97D, E97Q, K12G/E97A, I170A, L230A, I170A/L230A, and P166A variants of this enzyme. The EVB simulations model the observed effect of the P166A mutation on protein structure. The E97A, E97Q, and E97D mutations of the conserved E97 side chain result in ≤1.0 kcal mol–1 decreases in the activation barrier for substrate deprotonation. The agreement between experimental and computed activation barriers is within ±1 kcal mol–1, with a strong linear correlation between ΔG and Δ for all 11 variants, with slopes β = 0.73 (R2 = 0.994) and β = 0.74 (R2 = 0.995) for the deprotonation of DHAP and GAP, respectively. These Brønsted-type correlations show that the amino acid side chains examined in this study function to reduce the standard-state Gibbs free energy of reaction for deprotonation of the weak α-carbonyl carbon acid substrate to form the enediolate phosphate reaction intermediate. TIM utilizes the cationic side chain of K12 to provide direct electrostatic stabilization of the enolate oxyanion, and the nonpolar side chains of P166, I170, and L230 are utilized for the construction of an active-site cavity that provides optimal stabilization of the enediolate phosphate intermediate relative to the carbon acid substrate.

    Fulltekst tilgjengelig fra 2020-09-11 23:29
  • 31.
    Kulkarni, Yashraj
    et al.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Computational physical organic chemistry using the empirical valence bond approach2019Inngår i: Advances in Physical Organic Chemistry, ISSN 0065-3160, E-ISSN 2162-5921, Vol. 53, s. 69-104Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    There has been growing interest in applying the empirical valence bond approach to a range of (bio)chemical problems, primarily to study enzymatic and non-enzymatic catalysis, but also to studying other processes such as excited state chemistry and reaction dynamics. Despite its apparent theoretical simplicity, this approach is a powerful computational tool that can be used to reproduce and rationalize a wide range of experimental observables, such as linear free energy relationships, kinetic isotope effects, and temperature effects on reaction rates. We provide here both a theoretical background for this approach, as well as highlighting several of its broad applications in computational physical organic chemistry.

    Fulltekst tilgjengelig fra 2022-01-01 23:47
  • 32.
    Kulkarni, Yashraj S.
    et al.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Liao, Qinghua
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Bylehn, Fabian
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab. UCL, Dept Chem Engn, Torrington Pl, London WC1E 7JE, England.
    Amyes, Tina L.
    SUNY Buffalo, Dept Chem, Buffalo, NY 14260 USA.
    Richard, John P.
    SUNY Buffalo, Dept Chem, Buffalo, NY 14260 USA.
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase2018Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, nr 11, s. 3854-3857Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have previously performed empirical valence bond calculations of the kinetic activation barriers, Delta G(calc) double dagger, for the deprotonation of complexes between TIM and the whole substrate glyceraldehyde-3-phosphate (GAP, Kulkarni et al. J. Am. Chem. Soc. 2017, 139, 10514-10525). We now extend this work to also study the deprotonation of the substrate pieces glycolaldehyde (GA) and GA.HPi [HPi = phosphite dianion]. Our combined calculations provide activation barriers, Delta G(calc)(double dagger) for the TIM-catalyzed deprotonation of GAP (12.9 +/- 0.8 kcal.mol(-1)), of the substrate piece GA (15.0 +/- 2.4 kcal.mol(-1)), and of the pieces GA.HP, (15.5 +/- 3.5 kcal.mol(-1)). The effect of bound dianion on Delta G(calc) double dagger is small (<= 2.6 kcal.mol(-1)), in comparison to the much larger 12.0 and 5.8 kcal.mol(-1) intrinsic phosphodianion and phosphite dianion binding energy utilized to stabilize the transition states for TIM-catalyzed deprotonation of GAP and GA. HP, respectively. This shows that the dianion binding energy is essentially fully expressed at our protein model for the Michaelis complex, where it is utilized to drive an activating change in enzyme conformation. The results represent an example of the synergistic use of results from experiments and calculations to advance our understanding of enzymatic reaction mechanisms.

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  • 33.
    Kutin, Yury
    et al.
    Max Planck Institute for Chemical Energy Conversion, Germany.
    Kositzki, Ramona
    Freie Universität Berlin, Germany.
    Branca, Rui M.M.
    Karolinska Institutet, Sweden.
    Srinivas, Vivek
    Stockholm University, Sweden.
    Lundin, Daniel
    Stockholm University, Sweden.
    Haumann, Michael
    Freie Universität Berlin, Germany.
    Högbom, Martin
    Stockholm University, Sweden.
    Cox, Nicholas
    Australian National University, Australia.
    Griese, Julia J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Department of Biochemistry and Biophysics, Stockholm University.
    Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins2019Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 294, nr 48, s. 18372-18386Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A heterobimetallic Mn/Fe cofactor is present in the R2 subunit of class Ic ribonucleotide reductases (R2c) and in R2-like ligand-binding oxidases (R2lox). Although the protein-derived metal ligands are the same in both groups of proteins, the connectivity of the two metal ions and the chemistry each cofactor performs are different: in R2c, a one-electron oxidant, the Mn/Fe dimer is linked by two oxygen bridges (μ-oxo/μ-hydroxo), whereas in R2lox, a two-electron oxidant, it is linked by a single oxygen bridge (μ-hydroxo) and a fatty acid ligand. Here, we identified a second coordination sphere residue that directs the divergent reactivity of the protein scaffold. We found that the residue that directly precedes the N-terminal carboxylate metal ligand is conserved as a glycine within the R2lox group but not in R2c. Substitution of the glycine with leucine converted the resting-state R2lox cofactor to an R2c-like cofactor, a μ-oxo/μ-hydroxo–bridged MnIII/FeIII dimer. This species has recently been observed as an intermediate of the oxygen activation reaction in WT R2lox, indicating that it is physiologically relevant. Cofactor maturation in R2c and R2lox therefore follows the same pathway, with structural and functional divergence of the two cofactor forms following oxygen activation. We also show that the leucine-substituted variant no longer functions as a two-electron oxidant. Our results reveal that the residue preceding the N-terminal metal ligand directs the cofactor's reactivity toward one- or two-electron redox chemistry, presumably by setting the protonation state of the bridging oxygens and thereby perturbing the redox potential of the Mn ion.

    Fulltekst (pdf)
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  • 34.
    Liao, Qinghua
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab. Forschungszentrum Julich, Inst Complex Syst, Struct Biochem ICS 6, D-52425 Julich, Germany.
    Owen, Michael C.
    Masaryk Univ, CEITEC Cent European Inst Technol, Brno 62500, Czech Republic;Forschungszentrum Julich, Inst Complex Syst, Struct Biochem ICS 6, D-52425 Julich, Germany.
    Bali, Sofia
    Forschungszentrum Julich, Inst Complex Syst, Struct Biochem ICS 6, D-52425 Julich, Germany;New Mexico State Univ, 1780 E Univ Ave, Las Cruces, NM 88003 USA.
    Barz, Bogdan
    Forschungszentrum Julich, Inst Complex Syst, Struct Biochem ICS 6, D-52425 Julich, Germany;Heinrich Heine Univ Dusseldorf, Inst Theoret & Computat Chem, D-40225 Dusseldorf, Germany.
    Strodel, Birgit
    Forschungszentrum Julich, Inst Complex Syst, Struct Biochem ICS 6, D-52425 Julich, Germany;Heinrich Heine Univ Dusseldorf, Inst Theoret & Computat Chem, D-40225 Dusseldorf, Germany.
    A beta under stress: the effects of acidosis, Cu2+-binding, and oxidation on amyloid beta-peptide dimers2018Inngår i: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 54, s. 7766-7769Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In light of the high affinity of Cu2+ for Alzheimer's A(1-42) and its ability to subsequently catalyze the formation of radicals, we examine the effects of Cu2+ binding, A oxidation, and an acidic environment on the conformational dynamics of the smallest A(1-42) oligomer, the A(1-42) dimer. Transition networks calculated from Hamiltonian replica exchange molecular dynamics (H-REMD) simulations reveal that the decreased pH considerably increased the -sheet content, whereas Cu2+ binding increased the exposed hydrophobic surface area, both of which can contribute to an increased oligomerization propensity and toxicity.

  • 35.
    Liao, Qinghua
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Pabis, Anna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Strodel, Birgit
    Forschungszentrum Julich, Julich, Germany; Heinrich Heine Univ Dusseldorf, Dusseldorf, Germany.
    Kamerlin, Shina Caroline Lynn
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Extending the Nonbonded Cationic Dummy Model to Account for Ion-Induced Dipole Interactions2017Inngår i: Journal of Physical Chemistry Letters, ISSN 1948-7185, E-ISSN 1948-7185, Vol. 8, nr 21, s. 5408-5414Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Modeling metalloproteins often requires classical molecular dynamics (MD) simulations in order to capture their relevant motions, which in turn necessitates reliable descriptions of the metal centers involved. One of the most successful approaches to date is provided by the "cationic dummy model", where the positive charge of the metal ion is transferred toward dummy particles that are bonded to the central metal ion in a predefined coordination geometry. While this approach allows for ligand exchange, and captures the correct electrostatics as demonstrated for different divalent metal ions, current dummy models neglect ion-induced dipole interactions. In the present work, we resolve this weakness by taking advantage of the recently introduced 12-6-4 type Lennard-Jones potential to include ion-induced dipole interactions. We revise our previous dummy model for Mg2+ and demonstrate that the resulting model can simultaneously reproduce the experimental solvation free energy and metal ligand distances without the need for artificial restraints or bonds. As ion-induced dipole interactions become particularly important for highly charged metal ions, we develop dummy models for the biologically relevant ions Al3+, Fe3+, and Cr3+. Finally, the effectiveness of our new models is demonstrated in MD simulations of several diverse (and highly challenging to simulate) metalloproteins.

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  • 36.
    Marsavelski, Aleksandra
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Rudjer Boskovic Inst, Div Organ Chem & Biochem, Computat Organ Chem & Biochem Grp, Bijenicka Cesta 54, Zagreb 10000, Croatia;Univ Zagreb, Fac Sci, Dept Chem, Horvatovac 102a, Zagreb 10000, Croatia.
    Petrovic, Dusan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Bauer, Paul
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. KTH Royal Inst Technol, Dept Biophys, SciLifeLab, S-10691 Stockholm, Sweden.
    Vianello, Robert
    Rudjer Boskovic Inst, Div Organ Chem & Biochem, Computat Organ Chem & Biochem Grp, Bijenicka Cesta 54, Zagreb 10000, Croatia.
    Kamerlin, Shina Caroline Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Empirical Valence Bond Simulations Suggest a Direct Hydride Transfer Mechanism for Human Diamine Oxidase2018Inngår i: ACS Omega, ISSN 2470-1343, Vol. 3, nr 4, s. 3665-3674Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Diamine oxidase (DAO) is an enzyme involved in the regulation of cell proliferation and the immune response. This enzyme performs oxidative deamination in the catabolism of biogenic amines, including, among others, histamine, putrescine, spermidine, and spermine. The mechanistic details underlying the reductive half-reaction of the DAO-catalyzed oxidative deamination which leads to the reduced enzyme cofactor and the aldehyde product are, however, still under debate. The catalytic mechanism was proposed to involve a prototropic shift from the substrateSchiff base to the product-Schiff base, which includes the ratelimiting cleavage of the C alpha-H bond by the conserved catalytic aspartate. Our detailed mechanistic study, performed using a combined quantum chemical cluster approach with empirical valence bond simulations, suggests that the rate-limiting cleavage of the C alpha-H bond involves direct hydride transfer to the topaquinone cofactor. a mechanism that does not involve the formation of a Schiff base. Additional investigation of the D373E and D373N variants supported the hypothesis that the conserved catalytic aspartate is indeed essential for the reaction; however, it does not appear to serve as the catalytic base, as previously suggested. Rather, the electrostatic contributions of the most significant residues (including D373), together with the proximity of the Cu2+ cation to the reaction site, lower the activation barrier to drive the chemical reaction.

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  • 37.
    Maurer, Dirk
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Enugala, Thilak Reddy
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Hamnevik, Emil
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC.
    Bauer, Paul
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Lüking, Malin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Hillier, Heidi
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC.
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Dobritzsch, Doreen
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC.
    Widersten, Mikael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC.
    Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol DehydrogenasesInngår i: Artikkel i tidsskrift (Annet vitenskapelig)
  • 38.
    Mebs, Stefan
    et al.
    Freie Universität Berlin.
    Srinivas, Vivek
    Stockholm University.
    Kositzki, Ramona
    Freie Universität Berlin.
    Griese, Julia J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Högbom, Martin
    Stockholm University.
    Haumann, Michael
    Freie Universität Berlin.
    Fate of oxygen species from O2 activation at dimetal cofactors in an oxidase enzyme revealed by 57Fe nuclear resonance X-ray scattering and quantum chemistry2019Inngår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1860, nr 12, artikkel-id 148060Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Oxygen (O2) activation is a central challenge in chemistry and catalyzed at prototypic dimetal cofactors in biological enzymes with diverse functions. Analysis of intermediates is required to elucidate the reaction paths of reductive O2 cleavage. An oxidase protein from the bacterium Geobacillus kaustophilus, R2lox, was used for aerobic in-vitro reconstitution with only 57Fe(II) or Mn(II) plus 57Fe(II) ions to yield [FeFe] or [MnFe] cofactors under various oxygen and solvent isotopic conditions including 16/18O and H/D exchange. 57Fe-specific X-ray scattering techniques were employed to collect nuclear forward scattering (NFS) and nuclear resonance vibrational spectroscopy (NRVS) data of the R2lox proteins. NFS revealed Fe/Mn(III)Fe(III) cofactor states and Mössbauer quadrupole splitting energies. Quantum chemical calculations of NRVS spectra assigned molecular structures, vibrational modes, and protonation patterns of the cofactors, featuring a terminal water (H2O) bound at iron or manganese in site 1 and a metal-bridging hydroxide (μOH−) ligand. A procedure for quantitation and correlation of experimental and computational NRVS difference signals due to isotope labeling was developed. This approach revealed that the protons of the ligands as well as the terminal water at the R2lox cofactors exchange with the bulk solvent whereas 18O from 18O2 cleavage is incorporated in the hydroxide bridge. In R2lox, the two water molecules from four-electron O2 reduction are released in a two-step reaction to the solvent. These results establish combined NRVS and QM/MM for tracking of iron-based oxygen activation in biological and chemical catalysts and clarify the reductive O2 cleavage route in an enzyme.

  • 39.
    Moraleda-Munoz, Aurelio
    et al.
    Univ Granada, Fac Ciencias, Dept Microbiol, Avda Fuentenueva S-N, E-18071 Granada, Spain.
    Marcos-Torres, Francisco Javier
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Univ Granada, Fac Ciencias, Dept Microbiol, Avda Fuentenueva S-N, E-18071 Granada, Spain.
    Perez, Juana
    Univ Granada, Fac Ciencias, Dept Microbiol, Avda Fuentenueva S-N, E-18071 Granada, Spain.
    Munoz-Dorado, Jose
    Univ Granada, Fac Ciencias, Dept Microbiol, Avda Fuentenueva S-N, E-18071 Granada, Spain.
    Metal-responsive RNA polymerase extracytoplasmic function (ECF) sigma factors2019Inngår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 112, nr 2, s. 385-398Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    In order to survive, bacteria must adapt to multiple fluctuations in their environment, including coping with changes in metal concentrations. Many metals are essential for viability, since they act as cofactors of indispensable enzymes. But on the other hand, they are potentially toxic because they generate reactive oxygen species or displace other metals from proteins, turning them inactive. This dual effect of metals forces cells to maintain homeostasis using a variety of systems to import and export them. These systems are usually inducible, and their expression is regulated by metal sensors and signal-transduction mechanisms, one of which is mediated by extracytoplasmic function (ECF) sigma factors. In this review, we have focused on the metal-responsive ECF sigma factors, several of which are activated by iron depletion (FecI, FpvI and PvdS), while others are activated by excess of metals such as nickel and cobalt (CnrH), copper (CarQ and CorE) or cadmium and zinc (CorE2). We focus particularly on their physiological roles, mechanisms of action and signal transduction pathways.

    Fulltekst (pdf)
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  • 40.
    Osterlund, Nicklas
    et al.
    Stockholm Univ, Dept Biochem & Biophys, Arrhenius Labs, S-10691 Stockholm, Sweden;Stockholm Univ, Dept Environm Sci & Analyt Chem, Arrhenius Labs, S-10691 Stockholm, Sweden.
    Kulkarni, Yashraj S.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Misiaszek, Agata D.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wallin, Cecilia
    Stockholm Univ, Dept Biochem & Biophys, Arrhenius Labs, S-10691 Stockholm, Sweden.
    Krueger, Dennis M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Liao, Qinghua
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Rad, Farshid Mashayekhy
    Stockholm Univ, Dept Environm Sci & Analyt Chem, Arrhenius Labs, S-10691 Stockholm, Sweden.
    Jarvet, Juri
    Stockholm Univ, Dept Biochem & Biophys, Arrhenius Labs, S-10691 Stockholm, Sweden;NICPB, EE-12618 Tallinn, Estonia.
    Strodel, Birgit
    Forschungszentrum Julich, Inst Complex Syst Struct Biochem, D-52425 Julich, Germany.
    Warmlander, Sebastian K. T. S.
    Stockholm Univ, Dept Biochem & Biophys, Arrhenius Labs, S-10691 Stockholm, Sweden.
    Ilag, Leopold L.
    Stockholm Univ, Dept Environm Sci & Analyt Chem, Arrhenius Labs, S-10691 Stockholm, Sweden.
    Kamerlin, Shina C. L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Graslund, Astrid
    Stockholm Univ, Dept Biochem & Biophys, Arrhenius Labs, S-10691 Stockholm, Sweden.
    Amyloid-beta Peptide Interactions with Amphiphilic Surfactants: Electrostatic and Hydrophobic Effects2018Inngår i: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 9, nr 7, s. 1680-1692Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The amphiphilic nature of the amyloid-beta (A beta) peptide associated with Alzheimer's disease facilitates various interactions with biomolecules such as lipids and proteins, with effects on both structure and toxicity of the peptide. Here, we investigate these peptide-amphiphile interactions by experimental and computational studies of A beta(1-40) in the presence of surfactants with varying physicochemical properties. Our findings indicate that electrostatic peptide-surfactant interactions are required for coclustering and structure induction in the peptide and that the strength of the interaction depends on the surfactant net charge. Both aggregation-prone peptide-rich coclusters and stable surfactant-rich coclusters can form. Only A beta(1-40) monomers, but not oligomers, are inserted into surfactant micelles in this surfactant-rich state. Surfactant headgroup charge is suggested to be important as electrostatic peptide-surfactant interactions on the micellar surface seems to be an initiating step toward insertion. Thus, no peptide insertion or change in peptide secondary structure is observed using a nonionic surfactant. The hydrophobic peptide-surfactant interactions instead stabilize the A beta monomer, possibly by preventing self-interaction between the peptide core and C terminus, thereby effectively inhibiting the peptide aggregation process. These findings give increased understanding regarding the molecular driving forces for A beta aggregation and the peptide interaction with amphiphilic biomolecules.

  • 41.
    Pabis, Anna
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Risso, Valeria A.
    Univ Granada, Fac Ciencias, Dept Quim Fis, E-18071 Granada, Spain..
    Sanchez-Ruiz, Jose M.
    Univ Granada, Fac Ciencias, Dept Quim Fis, E-18071 Granada, Spain..
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Cooperativity and flexibility in enzyme evolution2018Inngår i: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 48, s. 83-92Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Enzymes are flexible catalysts, and there has been substantial discussion about the extent to which this flexibility contributes to their catalytic efficiency. What has been significantly less discussed is the extent to which this flexibility contributes to their evolvability. Despite this, recent years have seen an increasing number of both experimental and computational studies that demonstrate that cooperativity and flexibility play significant roles in enzyme innovation. This review covers key developments in the field that emphasize the importance of enzyme dynamics not just to the evolution of new enzyme function(s), but also as a property that can be harnessed in the design of new artificial enzymes.

    Fulltekst (pdf)
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  • 42.
    Petrović, Dušan
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Forschungszentrum Jülich, Inst Complex Syst Struct Biochem, Jülich, Germany.
    Bokel, Ansgar
    Heinrich Heine Univ Düsseldorf, Inst Biochem, Düsseldorf, Germany.
    Allan, Matthew
    Forschungszentrum Jülich, Inst Complex Syst Struct Biochem, Jülich, Germany; Penn State Univ, Schreyer Honors Coll, PA USA.
    Urlacher, Vlada B.
    Heinrich Heine Univ Düsseldorf, Inst Biochem, Düsseldorf, Germany.
    Strodel, Birgit
    Forschungszentrum Jülich, Inst Complex Syst Struct Biochem, Jülich, Germany; Heinrich Heine Univ Düsseldorf, Inst Theoret & Computat Chem, Düsseldorf, Germany.
    Simulation-Guided Design of Cytochrome P450 for Chemo- and Regioselective Macrocyclic Oxidation2018Inngår i: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 58, nr 4, s. 848-858Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Engineering high chemo-, regio-, and stereoselectivity is a prerequisite for enzyme usage in organic synthesis. Cytochromes P450 can oxidize a broad range of substrates, including macrocycles, which are becoming popular scaffolds for therapeutic agents. However, a large conformational space explored by macrocycles not only reduces the selectivity of oxidation but also impairs computational enzyme design strategies based on docking and molecular dynamics (MD) simulations. We present a novel design workflow that uses enhanced-sampling Hamiltonian replica exchange (HREX) MD and focuses on quantifying the substrate binding for suggesting the mutations to be made. This computational approach is applied to P450 BM3 with the aim to shift regioselectively toward one of the numerous possible positions during beta-cembrenediol oxidation. The predictions are experimentally tested and the resulting product distributions validate our design strategy, as single mutations led up to 5-fold regioselectivity increases. We thus conclude that the HREX-MD-based workflow is a promising tool for the identification of positions for mutagenesis aiming at P450 enzymes with improved regioselectivity.

  • 43.
    Stern, Ana Laura
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Van der Verren, Sander Egbert
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Kanchugal Puttaswamy, Sandesh
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Näsvall, Joakim
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Gutiérrez-de-Terán, Hugo
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräkningsbiologi och bioinformatik.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Structural mechanism of AadA, a dual specificity aminoglycoside adenylyltransferase from Salmonella enterica2018Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, s. 11481-11490Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3’’)(9) adenylyl transferase that O-adenylates position 3” of streptomycin and position 9 of spectinomycin. We previously reported the apo AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of wildtype AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site, and, in agreement with mutational data, critically interacts with fewer residues. Using structure-guided sequence analyses of ANT(3”)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the non-adenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.

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    fulltext
  • 44.
    Stsiapanava, Alena
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Crystal structure of ErmE-23S rRNA methyltransferase in macrolide resistance2019Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, nr 1, artikkel-id 14607Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pathogens often receive antibiotic resistance genes through horizontal gene transfer from bacteria that produce natural antibiotics. ErmE is a methyltransferase (MTase) from Saccharopolyspora erythraea that dimethylates A2058 in 23S rRNA using S-adenosyl methionine (SAM) as methyl donor, protecting the ribosomes from macrolide binding. To gain insights into the mechanism of macrolide resistance, the crystal structure of ErmE was determined to 1.75 Å resolution. ErmE consists of an N-terminal Rossmann-like α/ß catalytic domain and a C-terminal helical domain. Comparison with ErmC' that despite only 24% sequence identity has the same function, reveals highly similar catalytic domains. Accordingly, superposition with the catalytic domain of ErmC' in complex with SAM suggests that the cofactor binding site is conserved. The two structures mainly differ in the C-terminal domain, which in ErmE contains a longer loop harboring an additional 310 helix that interacts with the catalytic domain to stabilize the tertiary structure. Notably, ErmE also differs from ErmC' by having long disordered extensions at its N- and C-termini. A C-terminal disordered region rich in arginine and glycine is also a present in two other MTases, PikR1 and PikR2, which share about 30% sequence identity with ErmE and methylate the same nucleotide in 23S rRNA.

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  • 45.
    Szałaj, Natalia
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi.
    Lu, Lu
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Benediktsdottir, Andrea
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi.
    Zamaratski, Edouard
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi.
    Cao, Sha
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Olanders, Gustav
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi.
    Hedgecock, Charles
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi.
    Karlen, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi.
    Erdélyi, Máté
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi.
    Hughes, Diarmaid
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Mowbray, Sherry L
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Brandt, Peter
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi.
    Boronic ester-linked macrocyclic lipopeptides as serine protease inhibitors targeting Escherichia coli type I signal peptidase.2018Inngår i: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 157, s. 1346-1360Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type I signal peptidase, with its vital role in bacterial viability, is a promising but underexploited antibacterial drug target. In the light of steadily increasing rates of antimicrobial resistance, we have developed novel macrocyclic lipopeptides, linking P2 and P1' by a boronic ester warhead, capable of inhibiting Escherichia coli type I signal peptidase (EcLepB) and exhibiting good antibacterial activity. Structural modifications of the macrocyclic ring, the peptide sequence and the lipophilic tail led us to 14 novel macrocyclic boronic esters. It could be shown that macrocyclization is well tolerated in terms of EcLepB inhibition and antibacterial activity. Among the synthesized macrocycles, potent enzyme inhibitors in the low nanomolar range (e.g. compound 42f, EcLepB IC50 = 29 nM) were identified also showing good antimicrobial activity (e.g. compound 42b, E. coli WT MIC = 16 μg/mL). The unique macrocyclic boronic esters described here were based on previously published linear lipopeptidic EcLepB inhibitors in an attempt to address cytotoxicity and hemolysis. We show herein that structural changes to the macrocyclic ring influence both the cytotoxicity and hemolytic activity suggesting that the P2 to P1' linker provide means for optimizing off-target effects. However, for the present set of compounds we were not able to separate the antibacterial activity and cytotoxic effect.

  • 46.
    Söderholm, Annika
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    The Importance of Being Promiscuous: Understanding enzyme function, specificity, and evolution through structure2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Enzymes are known to be amazingly specific and efficient catalysts. However, many enzymes also have so-called promiscuous functions, i.e., they are able to catalyze other reactions than their main one. The promiscuous activities are often low, serendipitous, and under neutral selection but if conditions arise that make them beneficial, they can play an important role in the evolution of new enzymes. In this thesis, I present three studies where we have characterized different enzyme families by structural and biochemical methods. The studies demonstrate the occurrence of enzyme promiscuity and its potential role in evolution and organismal adaptation.

    In the first study, I describe the characterization of wild type and mutant HisA enzymes from Salmonella enterica. In the first part of this study, we could clarify the mechanistic cycle of HisA by solving crystal structures that showed different conformations of wild type HisA in complex with its labile substrate ProFAR (N´-[(5´-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide). In the second part of this study, structures of mutant enzymes from a real-time evolution study provided us with an atomic-level description of how HisA had evolved a new function. The HisA mutants had acquired TrpF activity, either in addition to (bifunctional generalists) or instead of (TrpF specialists) their HisA activity. In the second study, I present the crystal structure and demonstrate promiscuous activity of the TrpC enzyme from Pseudomonas aeruginosa. The activity data demonstrates that the enzyme can turn over a substrate that lacks a substituent that was previously considered essential for catalysis. In the third study, I present the structural and functional characterization of SAM (S-Adenosyl methionine) hydrolases from bacteriophages. These enzymes were discovered because of their ability to rescue auxotrophic bacteria by inducing expression of a promiscuous bacterial enzyme.  

    Delarbeid
    1. Structure of a phage-encoded SAM hydrolase enzyme provides insights in substrate binding and catalysis
    Åpne denne publikasjonen i ny fane eller vindu >>Structure of a phage-encoded SAM hydrolase enzyme provides insights in substrate binding and catalysis
    Vise andre…
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    Emneord
    SAM hydrolase, phage enzyme
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-366689 (URN)
    Tilgjengelig fra: 2018-11-22 Laget: 2018-11-22 Sist oppdatert: 2020-01-29
    2. Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA
    Åpne denne publikasjonen i ny fane eller vindu >>Two-step Ligand Binding in a (βα)8 Barrel Enzyme: Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA
    Vise andre…
    2015 (engelsk)Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 41, s. 24657-24668Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

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

    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-260701 (URN)10.1074/jbc.M115.678086 (DOI)000362598300003 ()26294764 (PubMedID)
    Forskningsfinansiär
    Swedish Research CouncilSwedish Foundation for Strategic Research EU, FP7, Seventh Framework Programme, 283570
    Tilgjengelig fra: 2015-08-23 Laget: 2015-08-23 Sist oppdatert: 2018-11-22
    3. Structural and functional innovations in the real-time evolution of new (βα)8 barrel enzymes
    Åpne denne publikasjonen i ny fane eller vindu >>Structural and functional innovations in the real-time evolution of new (βα)8 barrel enzymes
    Vise andre…
    2017 (engelsk)Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, nr 8, s. 4727-4732Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (βα)8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (βα)8 barrel as an inherently evolvable protein scaffold.

    Emneord
    HisA, TrpF, adaptive evolution, enzyme performance threshold
    HSV kategori
    Forskningsprogram
    Biologi med inriktning mot strukturbiologi; Biokemi; Biologi med inriktning mot molekylär evolution
    Identifikatorer
    urn:nbn:se:uu:diva-320223 (URN)10.1073/pnas.1618552114 (DOI)000400358000052 ()
    Tilgjengelig fra: 2017-04-18 Laget: 2017-04-18 Sist oppdatert: 2018-11-22bibliografisk kontrollert
    4. A bacteriophage enzyme induces bacterial metabolic perturbation that confers a novel promiscuous function
    Åpne denne publikasjonen i ny fane eller vindu >>A bacteriophage enzyme induces bacterial metabolic perturbation that confers a novel promiscuous function
    Vise andre…
    2018 (engelsk)Inngår i: Nature Ecology & Evolution, E-ISSN 2397-334X, Vol. 2, nr 8, s. 1321-1330Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    One key concept in the evolution of new functions is the ability of enzymes to perform promiscuous side-reactions that serve as a source of novelty that may become beneficial under certain conditions. Here, we identify a mechanism where a bacteriophage-encoded enzyme introduces novelty by inducing expression of a promiscuous bacterial enzyme. By screening for bacteriophage DNA that rescued an auxotrophic Escherichia coli mutant carrying a deletion of the ilvA gene, we show that bacteriophage-encoded S-adenosylmethionine (SAM) hydrolases reduce SAM levels. Through this perturbation of bacterial metabolism, expression of the promiscuous bacterial enzyme MetB is increased, which in turn complements the absence of IlvA. These results demonstrate how foreign DNA can increase the metabolic capacity of bacteria, not only by transfer of bona fide new genes, but also by bringing cryptic bacterial functions to light via perturbations of cellular physiology.

    sted, utgiver, år, opplag, sider
    Nature Publishing Group, 2018
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-355286 (URN)10.1038/s41559-018-0568-5 (DOI)000439505600024 ()
    Forskningsfinansiär
    Swedish Research CouncilKnut and Alice Wallenberg Foundation
    Tilgjengelig fra: 2018-06-27 Laget: 2018-06-27 Sist oppdatert: 2018-11-22bibliografisk kontrollert
    5. Structure and substrate ambiguity of TrpC from Pseudomonas aeruginosa
    Åpne denne publikasjonen i ny fane eller vindu >>Structure and substrate ambiguity of TrpC from Pseudomonas aeruginosa
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    The enzyme TrpC catalyzes the formation of Indole-3-glycerol phosphate (IGP) from 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate as part of the tryptophan biosynthesis pathway. The reaction mechanism follows a series of condensation, decarboxylation, and dehydration. The decarboxylation has been assumed to constitute an essential step of the mechanism since no activity with decarboxylated substrate was observed in an early study on the TrpC:TrpF fusion protein from Escherichia coli (Smith 1962). Here, we refute this assumption by demonstrating IGP formation catalyzed by both TrpC from Pseudomonas aeruginosa and from E.coli. We show that P. aeruginosa TrpC is more active on decarboxylated substrate than E.coli TrpC and, by solving the crystal structure of P. aeruginosa TrpC, we provide structure-based hypotheses on their difference in promiscuous activity.

    Emneord
    TrpC, IGPS
    HSV kategori
    Forskningsprogram
    Biokemi
    Identifikatorer
    urn:nbn:se:uu:diva-366660 (URN)
    Tilgjengelig fra: 2018-11-22 Laget: 2018-11-22 Sist oppdatert: 2018-11-30
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  • 47.
    Söderholm, Annika
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Guo, Xiaohu
    Kanchugal Puttaswamy, Sandesh
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Eckhard, Ulrich
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Warsi, Omar M.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Jerlstrom-Hultqvist, Joel
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Andersson, Dan I
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Structure of a phage-encoded SAM hydrolase enzyme provides insights in substrate binding and catalysisManuskript (preprint) (Annet vitenskapelig)
  • 48.
    Söderholm, Annika
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    Selmer, Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Newton, Matilda
    Patrick, Wayne
    Structure and substrate ambiguity of TrpC from Pseudomonas aeruginosaManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    The enzyme TrpC catalyzes the formation of Indole-3-glycerol phosphate (IGP) from 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate as part of the tryptophan biosynthesis pathway. The reaction mechanism follows a series of condensation, decarboxylation, and dehydration. The decarboxylation has been assumed to constitute an essential step of the mechanism since no activity with decarboxylated substrate was observed in an early study on the TrpC:TrpF fusion protein from Escherichia coli (Smith 1962). Here, we refute this assumption by demonstrating IGP formation catalyzed by both TrpC from Pseudomonas aeruginosa and from E.coli. We show that P. aeruginosa TrpC is more active on decarboxylated substrate than E.coli TrpC and, by solving the crystal structure of P. aeruginosa TrpC, we provide structure-based hypotheses on their difference in promiscuous activity.

  • 49.
    Uduwela, Dimanthi R.
    et al.
    Australian Natl Univ, Res Sch Chem, Canberra, ACT 2601, Australia.
    Pabis, Anna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär biofysik.
    Stevenson, Bradley J.
    Australian Natl Univ, Res Sch Chem, Canberra, ACT 2601, Australia.
    Kamerlin, Shina C. Lynn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi.
    McLeod, Malcolm D.
    Australian Natl Univ, Res Sch Chem, Canberra, ACT 2601, Australia.
    Enhancing the Steroid Sulfatase Activity of the Arylsulfatase from Pseudomonas aeruginosa2018Inngår i: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 8, nr 9, s. 8902-8914Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Steroidal sulfate esters play a central role in many physiological processes. They serve as the reservoir for endogenous sex hormones and form a significant fraction of the steroid metabolite pool. The analysis of steroid sulfates is thus essential in fields such as medical science and sports drug testing. Although the direct detection of steroid sulfates can be readily achieved using liquid chromatography-mass spectrometry, many analytical approaches, including gas chromatography-mass spectrometry, are hampered due to the lack of suitable enzymatic or chemical methods for sulfate ester hydrolysis prior to analysis. Enhanced methods of steroid sulfate hydrolysis would expand analytical possibilities for the study of these widely occurring metabolites. The arylsulfatase from Pseudomonas aeruginosa (PaS) is a purified enzyme capable of hydrolyzing steroid sulfates. However, this enzyme requires improvement to hydrolytic activity and substrate scope in order to be useful in analytical applications. These improvements were sought by applying semirational design to mutate amino acid residues neighboring the enzyme active site. Mutagenesis was implemented on both single and multiple residue sites. Screening by ultra-high performance liquid chromatography-mass spectrometry was performed to test the steroid sulfate hydrolysis activity of these mutant libraries against testosterone sulfate. This approach revealed the steroid sulfate binding pocket and resulted in three mutants that showed an improvement in catalytic efficiency (V-max/K-M) of more than 150 times that of wild-type PaS. The substrate scope of PaS was expanded, and a modest increase in thermostability was observed. Finally, molecular dynamics simulations of enzyme-substrate complexes were used to provide qualitative insight into the structural origin of the observed effects.

  • 50.
    Xu, Hongyi
    et al.
    Stockholm University.
    Lebrette, Hugo
    Stockholm University.
    Clabbers, Max T. B.
    Stockholm University.
    Zhao, Jingjing
    Stockholm University.
    Griese, Julia J.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Strukturbiologi. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden..
    Zou, Xiaodong
    Stockholm University.
    Högbom, Martin
    Stockholm University.
    Solving a new R2lox protein structure by microcrystal electron diffraction2019Inngår i: Science Advances, E-ISSN 2375-2548, Vol. 5, nr 8, artikkel-id eaax4621Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Microcrystal electron diffraction (MicroED) has recently shown potential for structural biology. It enables the study of biomolecules from micrometer-sized 3D crystals that are too small to be studied by conventional x-ray crystallography. However, to date, MicroED has only been applied to redetermine protein structures that had already been solved previously by x-ray diffraction. Here, we present the first new protein structure—an R2lox enzyme—solved using MicroED. The structure was phased by molecular replacement using a search model of 35% sequence identity. The resulting electrostatic scattering potential map at 3.0-Å resolution was of sufficient quality to allow accurate model building and refinement. The dinuclear metal cofactor could be located in the map and was modeled as a heterodinuclear Mn/Fe center based on previous studies. Our results demonstrate that MicroED has the potential to become a widely applicable tool for revealing novel insights into protein structure and function.

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