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  • 1.
    Amrein, Beat Anton
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Runthala, Ashish
    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.
    In Silico-Directed Evolution Using CADEE2018In: Computational Methods in Protein Evolution / [ed] T. Tobias Sikosek, Springer Science+Business Media, LLC, part of Springer Nature , 2018, p. 381-415Chapter in book (Other academic)
  • 2.
    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.

  • 3.
    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.

  • 4.
    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.
    Aqvist, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. 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, Science for Life Laboratory, SciLifeLab. 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: Software Quality Professional, ISSN 1522-0540, E-ISSN 2352-7110, SoftwareX, ISSN 2352-7110Article 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.

  • 5.
    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.

  • 6.
    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.

  • 7.
    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.

  • 8.
    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).

  • 9.
    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.

  • 10.
    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.

  • 11.
    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)
  • 12.
    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)
  • 13.
    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).

  • 14.
    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.

    The full text will be freely available from 2019-06-01 19:18
  • 15.
    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.

  • 16.
    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.

  • 17.
    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.

  • 18.
    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.

  • 19.
    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.

  • 20.
    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)
  • 21.
    Pabis, Anna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    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 University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Cooperativity and flexibility in enzyme evolution2018In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 48, p. 83-92Article in journal (Refereed)
    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.

  • 22.
    Petrović, Dušan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. 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 Oxidation2018In: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 58, no 4, p. 848-858Article in journal (Refereed)
    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.

  • 23.
    Stern, Ana Laura
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Van der Verren, Sander Egbert
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kanchugal, Sandesh Puttaswamy
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Näsvall, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Structural mechanism of AadA, a dual specificity aminoglycoside adenylyltransferase from Salmonella enterica2018In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, p. 11481-11490Article in journal (Refereed)
    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.

  • 24.
    Szałaj, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Lu, Lu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Benediktsdottir, Andrea
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Zamaratski, Edouard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Olanders, Gustav
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Hedgecock, Charles
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Karlen, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Erdélyi, Máté
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mowbray, Sherry L
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Boronic ester-linked macrocyclic lipopeptides as serine protease inhibitors targeting Escherichia coli type I signal peptidase.2018In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 157, p. 1346-1360Article in journal (Refereed)
    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.

  • 25.
    Söderholm, Annika
    et al.
    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.
    Guo, Xiaohu
    Kanchugal, Sandesh Puttaswamy
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Eckhard, Ulrich
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Warsi, Omar M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jerlstrom-Hultqvist, Joel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Structure of a phage-encoded SAM hydrolase enzyme provides insights in substrate binding and catalysisManuscript (preprint) (Other academic)
  • 26.
    Söderholm, Annika
    et al.
    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.
    Newton, Matilda
    Patrick, Wayne
    Structure and substrate ambiguity of TrpC from Pseudomonas aeruginosaManuscript (preprint) (Other academic)
    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.

  • 27.
    Uduwela, Dimanthi R.
    et al.
    Australian Natl Univ, Res Sch Chem, Canberra, ACT 2601, Australia.
    Pabis, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Stevenson, Bradley J.
    Australian Natl Univ, Res Sch Chem, Canberra, ACT 2601, Australia.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    McLeod, Malcolm D.
    Australian Natl Univ, Res Sch Chem, Canberra, ACT 2601, Australia.
    Enhancing the Steroid Sulfatase Activity of the Arylsulfatase from Pseudomonas aeruginosa2018In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 8, no 9, p. 8902-8914Article in journal (Refereed)
    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.

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