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  • 1.
    Barrozo, Alexandre
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Liao, Qinghua
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Esguerra, Mauricio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Marloie, Gael
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Computer simulations of the catalytic mechanism of wild-type and mutant beta-phosphoglucomutase2018In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 16, no 12, p. 2060-2073Article in journal (Refereed)
    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.

  • 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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. 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 Shape2018In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, no 1, p. 319-327Article in journal (Refereed)
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Amrein, Beat Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Esguerra, Mauricio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Q6: A comprehensive toolkit for empirical valence bond and related free energy calculations2018In: SoftwareX, ISSN 2352-7110, p. 388-395Article in journal (Refereed)
    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.

  • 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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    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 FimH2018In: Beilstein Journal of Organic Chemistry, ISSN 2195-951X, E-ISSN 1860-5397, Vol. 14, p. 1890-1900Article in journal (Refereed)
    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.

  • 5. Ben-David, Moshe
    et al.
    Soskine, Misha
    Dubovetskyi, Artem
    Cherukuri, Kesava-Phaneendra
    Dym, Orly
    Sussman, Joel L
    Liao, Qinghua
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Szeler, Klaudia
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kamerlin, Shina C. Lynn
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Tawfik, Dan S
    Enzyme Evolution An Epistatic Ratchet versus a Smooth Reversible Transition2019In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 37, no 4, p. 1133-1147Article in journal (Refereed)
    Abstract [en]

    Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.

  • 6.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Corbella, Marina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - 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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    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 Isomerases2019In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 9, no 9, p. 8388-8396Article in journal (Refereed)
    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.

  • 7.
    Durall de la Fuente, Claudia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Kanchugal Puttaswamy, Sandesh
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Phosphoenolpyruvate carboxylase in Synechococcus PCC 7002: Oligomerization, structure, and characteristics2020In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 10, article id 3607Article in journal (Refereed)
  • 8.
    Elison Kalman, Grim
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Purification, functional characterization and crystallization of the PerR peroxide sensor from Saccharopolyspora erythraea2019Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    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.

  • 9.
    Ge, Xueliang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Oliveira, Ana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Inhibition of translation termination by small molecules targeting ribosomal release factors2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 15424Article in journal (Refereed)
    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.

  • 10.
    Griese, Julia J.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. 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 oxidase2018In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 23, no 6, p. 879-886Article in journal (Refereed)
    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.

  • 11.
    Griese, Julia J.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. 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-XAD2019In: Acta Crystallographica Section D: Structural Biology, ISSN 2059-7983, Vol. 75, no 8, p. 764-771Article in journal (Refereed)
    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.

  • 12.
    Griese, Julia J.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. 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 oxidase2019In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 24, no 2, p. 211-221Article in journal (Refereed)
    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.

  • 13.
    Grāve, Kristīne
    et al.
    Stockholm University.
    Lambert, Wietske
    PRA Health Sciences, Assen, The Netherlands.
    Berggren, Gustav
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Griese, Julia J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. 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 access2019In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 24, no 6, p. 849-861Article in journal (Refereed)
    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.

  • 14.
    Harish, Ajith
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    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"2018In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 149, p. 137-138Article in journal (Other academic)
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Kurland, Charles G.
    Lund Univ, Sect Microbial Ecol, Dept Biol, Lund, Sweden..
    Mitochondria are not captive bacteria2017In: Journal of Theoretical Biology, ISSN 0022-5193, E-ISSN 1095-8541, Vol. 434, p. 88-98Article in journal (Refereed)
    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 University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme2018In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 5, no 3, p. 269-282Article in journal (Refereed)
    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.

  • 17.
    Jerlström-Hultqvist, Joel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
    Warsi, Omar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Söderholm, Annika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Knopp, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eckhard, Ulrich
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Vorontsov, Egor
    Proteomics Core Facility at Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A bacteriophage enzyme induces bacterial metabolic perturbation that confers a novel promiscuous function2018In: Nature Ecology & Evolution, E-ISSN 2397-334X, Vol. 2, no 8, p. 1321-1330Article in journal (Refereed)
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Of spiders, bugs, and men: Structural and functional studies of proteins involved in assembly2018Doctoral thesis, comprehensive summary (Other academic)
    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.

    List of papers
    1. The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite
    Open this publication in new window or tab >>The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite
    Show others...
    2014 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 7, p. 1809-1816Article in journal (Refereed) 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.

    Keywords
    CysZ, Sulfate, Transport, Membrane protein, Inhibition
    National Category
    Biochemistry and Molecular Biology Biophysics
    Identifiers
    urn:nbn:se:uu:diva-227987 (URN)10.1016/j.bbamem.2014.03.003 (DOI)000336695300014 ()
    Available from: 2014-07-04 Created: 2014-07-02 Last updated: 2018-11-23Bibliographically approved
    2. Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis
    Open this publication in new window or tab >>Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis
    2018 (English)In: Acta crystallographica. Section D, Structural biology, ISSN 2059-7983, Vol. 74, no Pt 11, p. 1053-1062Article in journal (Refereed) 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.

    Keywords
    Proteus mirabilis, adhesins, fimbriae, urinary-tract infection
    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-366693 (URN)10.1107/S2059798318012391 (DOI)000449044300003 ()30387764 (PubMedID)
    Funder
    Swedish Research Council
    Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2019-01-07Bibliographically approved
    3. Structural basis for MrpH-dependent Proteus mirabilis biofilm formation
    Open this publication in new window or tab >>Structural basis for MrpH-dependent Proteus mirabilis biofilm formation
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    (English)Manuscript (preprint) (Other academic)
    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).

    Keywords
    fimbriae; adhesins; biofilm; urinary tract infection; Proteus mirabilis
    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-366696 (URN)
    Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-23
    4. Conversion of spidroin dope to spider silk involves an asymmetric dimer intermediate
    Open this publication in new window or tab >>Conversion of spidroin dope to spider silk involves an asymmetric dimer intermediate
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Structural Biology
    Identifiers
    urn:nbn:se:uu:diva-366699 (URN)
    Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-23
    5. Bri2 BRICHOS domain binds Bri23 and depends on conserved face A tyrosine residues for anti-amyloid activity
    Open this publication in new window or tab >>Bri2 BRICHOS domain binds Bri23 and depends on conserved face A tyrosine residues for anti-amyloid activity
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Keywords
    Bri2, BRICHOS, amyloid, Alzheimer's disease, molecular chaperone
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-366698 (URN)
    Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-23
  • 19.
    Jiang, Wangshu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Askarieh, Glareh
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Shkumatov, Alexander
    Structural Biology Brussels, Belgium.
    Hedhammar, My
    KTH royal institute of technology.
    Knight, Stefan D
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Conversion of spidroin dope to spider silk involves an asymmetric dimer intermediateManuscript (preprint) (Other academic)
  • 20.
    Jiang, Wangshu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Askarieh, Glareh
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    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 intermediate2019In: ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, ISSN 2059-7983, Vol. 75, p. 618-627Article in journal (Refereed)
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Bri2 BRICHOS domain binds Bri23 and depends on conserved face A tyrosine residues for anti-amyloid activityManuscript (preprint) (Other academic)
  • 22.
    Jiang, Wangshu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Ubhayasekera, Wimal
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Structural basis for MrpH-dependent Proteus mirabilis biofilm formationManuscript (preprint) (Other academic)
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Ubhayasekera, Wimal
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Pearson, Melanie M.
    Univ Michigan, Sch Med, Dept Microbiol & Immunol, Ann Arbor, MI USA.
    Knight, Stefan D.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis2018In: Acta crystallographica. Section D, Structural biology, ISSN 2059-7983, Vol. 74, no Pt 11, p. 1053-1062Article in journal (Refereed)
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Steffen-Munsberg, Fabian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    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 ancestor2018In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, no 6, p. 548-555Article in journal (Refereed)
    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.

  • 25.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    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 channel2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 21, p. E4900-E4909Article in journal (Refereed)
    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.

  • 26.
    Kulkarni, Yashraj
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    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 University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    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 Isomerase2019In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 141, no 40, p. 16139-16150Article in journal (Refereed)
    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.

    The full text will be freely available from 2020-09-11 23:29
  • 27.
    Kulkarni, Yashraj
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C. Lynn
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Computational physical organic chemistry using the empirical valence bond approach2019In: Advances in Physical Organic Chemistry, ISSN 0065-3160, E-ISSN 2162-5921, Vol. 53, p. 69-104Article in journal (Refereed)
    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.

    The full text will be freely available from 2022-01-01 23:47
  • 28.
    Kulkarni, Yashraj S.
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Liao, Qinghua
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bylehn, Fabian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, 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 University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase2018In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, no 11, p. 3854-3857Article in journal (Refereed)
    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.

  • 29.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Department of Biochemistry and Biophysics, Stockholm University.
    Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins2019In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 294, no 48, p. 18372-18386Article in journal (Refereed)
    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.

  • 30.
    Liao, Qinghua
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, 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 dimers2018In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 54, p. 7766-7769Article in journal (Refereed)
    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.

  • 31.
    Liao, Qinghua
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pabis, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Strodel, Birgit
    Forschungszentrum Julich, Julich, Germany; Heinrich Heine Univ Dusseldorf, Dusseldorf, Germany.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Extending the Nonbonded Cationic Dummy Model to Account for Ion-Induced Dipole Interactions2017In: Journal of Physical Chemistry Letters, ISSN 1948-7185, E-ISSN 1948-7185, Vol. 8, no 21, p. 5408-5414Article in journal (Refereed)
    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.

  • 32.
    Marsavelski, Aleksandra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. 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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. 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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Empirical Valence Bond Simulations Suggest a Direct Hydride Transfer Mechanism for Human Diamine Oxidase2018In: ACS Omega, ISSN 2470-1343, Vol. 3, no 4, p. 3665-3674Article in journal (Refereed)
    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.

  • 33.
    Maurer, Dirk
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Hamnevik, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lüking, Malin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Hillier, Heidi
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol DehydrogenasesIn: Article in journal (Other academic)
  • 34.
    Mebs, Stefan
    et al.
    Freie Universität Berlin.
    Srinivas, Vivek
    Stockholm University.
    Kositzki, Ramona
    Freie Universität Berlin.
    Griese, Julia J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    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 chemistry2019In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1860, no 12, article id 148060Article in journal (Refereed)
    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.

  • 35.
    Moraleda-Munoz, Aurelio
    et al.
    Univ Granada, Fac Ciencias, Dept Microbiol, Avda Fuentenueva S-N, E-18071 Granada, Spain.