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

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

  • 2.
    Andaloussi, Mounir
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Henriksson, Lena M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wieckowska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Lindh, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Larsson, Anna M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Suresh, Surisetti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Iyer, Harini
    Srinivasa, Bachally R.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase2011In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 14, p. 4964-4976Article in journal (Refereed)
    Abstract [en]

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

  • 3.
    Andersson, C. Evalena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mowbray, Sherry L.
    Activation of Ribokinase by Monovalent Cations2002In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 315, no 3, p. 409-419Article in journal (Refereed)
  • 4.
    Andersson, C. Evalena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Simon, Jill A.
    Cameron, Alexander D.
    Mowbray, Sherry L.
    Specificity and Activity of Escherichia coli RibokinaseManuscript (Other academic)
  • 5.
    Andersson, C. Evalena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Simon, Jill A.
    Cameron, Alexander D.
    Mowbray, Sherry L.
    Specificity and Activity of Escherichia coli RibokinaseManuscript (Other academic)
  • 6.
    Arand, M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. PROGRAM IN STRUCTURAL MOLECULAR BIOLOGY.
    Cronin, A
    Oesch, F
    Mowbray, Sherry L
    Department of Molecular Biosciences, Swedish University of Agricultural Sciences.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    The telltale structures of epoxide hydrolases2003In: Drug metabolism reviews (Softcover ed.), ISSN 0360-2532, E-ISSN 1097-9883, Vol. 35, no 4, p. 365-383Article in journal (Refereed)
    Abstract [en]

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

  • 7. Arand, Michael
    et al.
    Hallberg, B Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Zou, Jinyu
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Oesch, Franz
    van der Werf, Mariette
    de Bont, Jan A M
    Jones, T Alwyn
    Mowbray, Sherry L
    Department of Molecular Biosciences, Swedish University of Agricultural Science.
    Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site.2003In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, no 11, p. 2583-2592Article in journal (Refereed)
  • 8.
    Björkelid, Christofer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Henriksson, Lena M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Stern, Ana Laura
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structural and functional studies of mycobacterial IspD enzymes2011In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 67, p. 403-414Article in journal (Refereed)
    Abstract [en]

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

  • 9.
    Björkelid, Christofer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-9000982012In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 68, p. 134-143Article in journal (Refereed)
    Abstract [en]

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

  • 10. Burgos, Emmanuel
    et al.
    Roos, Annette K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mowbray, Sherry L.
    Salmon, Laurent
    Synthesis of 5-deoxy-5-phospho-D-ribonohydroxamic acid : a new competitive and selective inhibitor of type b ribose-5-phosphate isomerase from Mycobacterium tuberculosis2005In: Tetrahedron letters, ISSN 0040-4039, Vol. 46, no 21, p. 3691-3694Article in journal (Refereed)
  • 11. Carta, Fabrizio
    et al.
    Maresca, Alfonso
    Covarrubias, Adrian Suarez
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mowbray, S.L.
    Jones, Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Supuran, Claudiu T.
    Carbonic anhydrase inhibitors. Characterization and inhibition studies of the most active beta-carbonic anhydrase from Mycobacterium tuberculosis, Rv3588c2009In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 19, no 23, p. 6649-6654Article in journal (Refereed)
    Abstract [en]

    The Rv3588c gene product of Mycobacterium tuberculosis, a beta-carbonic anhydrase (CA, EC 4.2.1.1) denominated here mtCA 2, shows the highest catalytic activity for CO2 hydration (k(cat) of 9.8 x 10(5) s(-1), and k(cat)/K-m of 9.3 x 10(7) M-1 s(1)) among the three beta-CAs encoded in the genome of this pathogen. A series of sulfonamides/sulfamates was assayed for their interaction with mtCA 2, and some diazenylbenzenesulfonamides were synthesized from sulfanilamide/metanilamide by diazotization followed by coupling with amines or phenols. Several low nanomolar mtCA 2 inhibitors have been detected among which acetazolamide, ethoxzolamide and some 4-diazenylbenzenesulfonamides (K(I)s of 9-59 nM). As the Rv3588c gene was shown to be essential to the growth of M. tuberculosis, inhibition of this enzyme may be relevant for the design of antituberculosis drugs possessing a novel mechanism of action.

  • 12.
    Chaudhuri, B.N.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ko, J
    Park, C
    Jones, T.A.
    Mowbray, S.L.
    Structure of D-allose binding protein from Escherichia coli bound to D-allose at 1.8 angstrom resolution1999In: JOURNAL OF MOLECULAR BIOLOGY, ISSN 0022-2836, Vol. 286, no 5, p. 1519-1531Article in journal (Refereed)
    Abstract [en]

    ABC transport systems for import or export of nutrients and other substances across the cell membrane are widely distributed in nature. In most bacterial systems, a periplasmic component is the primary determinant of specificity of the transport complex a

  • 13. Chofor, Rene
    et al.
    Sooriyaarachchi, Sanjeewani
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Risseeuw, Martijn D. P.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pouyez, Jenny
    Johny, Chinchu
    Haymond, Amanda
    Everaert, Annelien
    Dowd, Cynthia S.
    Maes, Louis
    Coenye, Tom
    Alex, Alexander
    Couch, Robin D.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wouters, Johan
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Van Calenbergh, Serge
    Synthesis and Bioactivity of beta-Substituted Fosmidomycin Analogues Targeting 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase2015In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 58, no 7, p. 2988-3001Article in journal (Refereed)
    Abstract [en]

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

  • 14.
    Covarrubias, Adrian Suarez
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Högbom, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Carroll, Paul
    Mannerstedt, Karin
    Oscarson, Stefan
    Parish, Tanya
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mowbray, Sherry
    Structural, biochemical and in vivo investigations of the threonine synthase from Mycobacterium tuberculosis2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 381, no 3, p. 622-633Article in journal (Refereed)
    Abstract [en]

    Threonine biosynthesis is a general feature of prokaryotes, eukaryotic microorganisms, and higher plants. Since mammals lack the appropriate synthetic machinery, instead obtaining the amino acid through their diet, the pathway is a potential focus for the development of novel antibiotics, antifungal agents, and herbicides. Threonine synthase (TS), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the final step in the pathway, in which L-homoserine phosphate and water are converted into threonine and inorganic phosphate. In the present publication, we report structural and functional studies of Mycobacterium tuberculosis TS, the product of the rv1295 (thrC) gene. The structure gives new insights into the catalytic mechanism of TSs in general, specifically by suggesting the direct involvement of the phosphate moiety of the cofactor, rather than the inorganic phosphate product, in transferring a proton from C4' to C-gamma in the formation of the alpha beta-unsaturated aldimine. It further provides a basis for understanding why this enzyme has a higher pH optimum than has been reported elsewhere for TSs and gives rise to the prediction that the equivalent enzyme from Thermus thermophilus will exhibit similar behavior. A deletion of the relevant gene generated a strain of M. tuberculosis that requires threonine for growth, such auxotrophic strains are frequently attenuated in vivo, indicating that TS is a potential drug target in this organism.

  • 15.
    De Rosa, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lu, Lu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Zamaratski, Edouard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Szałaj, Natalia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wadensten, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lenhammar, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Roos, Annette K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Karlen, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)2017In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 3, p. 897-911Article in journal (Refereed)
    Abstract [en]

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

  • 16.
    Ericsson, Daniel J.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Kasrayan, Alex
    Johansson, Patrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Sandström, Anders G.
    Bäckvall, Jan-Erling
    Mowbray, Sherry L.
    Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala, Sweden.
    X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, no 1, p. 109-119Article in journal (Refereed)
    Abstract [en]

    In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the X-ray structure of lipase A from Candida antarctica, solved by single isomorphous replacement with anomalous scattering, and refined to 2.2-A resolution. The structure is the first from a novel family of lipases. Contrary to previous predictions, the fold includes a well-defined lid as well as a classic alpha/beta hydrolase domain. The catalytic triad is identified as Ser184, Asp334 and His366, which follow the sequential order considered to be characteristic of lipases; the serine lies within a typical nucleophilic elbow. Computer docking studies, as well as comparisons to related structures, place the carboxylate group of a fatty acid product near the serine nucleophile, with the long lipid tail closely following the path through the lid that is marked by a fortuitously bound molecule of polyethylene glycol. For an ester substrate to bind in an equivalent fashion, loop movements near Phe431 will be required, suggesting the primary focus of the conformational changes required for interfacial activation. Such movements will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation.

  • 17.
    FLOCCO, MM
    et al.
    Uppsala University.
    MOWBRAY, SL
    Uppsala University.
    C-ALPHA-BASED TORSION ANGLES - A SIMPLE TOOL TO ANALYZE PROTEIN CONFORMATIONAL-CHANGES1995In: PROTEIN SCIENCE, ISSN 0961-8368, Vol. 4, no 10, p. 2118-2122Article in journal (Other academic)
    Abstract [en]

    A simple method is presented for the analysis of protein conformational changes based on the comparison of torsion angles defined by four consecutive C alpha atoms. The technique was applied successfully to proteins that undergo hinge motion and shear mot

  • 18.
    FLOCCO, MM
    et al.
    Uppsala University.
    MOWBRAY, SL
    Uppsala University.
    STRANGE BEDFELLOWS - INTERACTIONS BETWEEN ACIDIC SIDE-CHAINS IN PROTEINS1995In: JOURNAL OF MOLECULAR BIOLOGY, ISSN 0022-2836, Vol. 254, no 1, p. 96-105Article in journal (Other academic)
    Abstract [en]

    The oxygen atoms of two acidic side-chains are frequently found within hydrogen-bonding distance of each other in proteins. Two distinct types of cases are common. In metal-binding sites, the oxygen atoms are brought near (average closest approach 3.0 Ang

  • 19.
    Gising, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Nilsson, Mikael T
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Odell, Luke R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Yahiaoui, Samir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Lindh, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Iyer, Harini
    AstraZeneca India.
    Sinha, Achyut M
    AstraZeneca India.
    Srinivasa, Bachally R
    AstraZeneca India.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Trisubstituted Imidazoles as Mycobacterium tuberculosis Glutamine Synthetase Inhibitors2012In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, no 6, p. 2894-2898Article in journal (Refereed)
    Abstract [en]

    Mycobacterium tuberculosis glutamine synthetase (MtGS) is a promising target for antituberculosis drug discovery. In a recent high-throughput screening study we identified several classes of MtGS inhibitors targeting the ATP-binding site. We now explore one of these classes, the 2-tert-butyl-4,5-diarylimidazoles, and present the design, synthesis, and X-ray crystallographic studies leading to the identification of MtGS inhibitors with submicromolar IC(50) values and promising antituberculosis MIC values.

  • 20.
    Grantcharova, N.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ubhayasekera, W.
    Mowbray, S.L.
    McCormick, J. R.
    Flärdh, K.
    A missense mutation in ftsZ differentially affects vegetative and developmentally controlled cell division in Streptomyces coelicolor2003In: Molecular Microbiology, Vol. 47, p. 645-656Article in journal (Refereed)
  • 21.
    Henriksson, Lena M
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mowbray, Sherry L
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    The 1.9 A resolution structure of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase, a potential drug target.2006In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 62, no Pt 7, p. 807-13Article in journal (Refereed)
    Abstract [en]

    1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzes the NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate, as the second step of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate pathway found in many bacteria and plants. The end product, isopentenyl diphosphate, is the precursor of various isoprenoids vital to all living organisms. The pathway is not found in humans; the mevalonate pathway is instead used for the formation of isopentenyl diphosphate. This difference, combined with its essentiality, makes the reductoisomerase an excellent drug target in a number of pathogenic organisms. The structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Mycobacterium tuberculosis (Rv2870c) was solved by molecular replacement and refined to a resolution of 1.9 A. The enzyme exhibited an estimated kcat of 5.3 s-1 and Km and kcat/Km values of 7.2 microM and 7.4x10(5) M-1 s-1 for NADPH and 340 microM and 1.6x10(4) M-1 s-1 for 1-deoxy-D-xylulose 5-phosphate. In the structure, a sulfate is bound at the expected site of the phosphate moiety of the sugar substrate. The M. tuberculosis enzyme displays a similar fold to the previously published structures from Escherichia coli and Zymomonas mobilis. Comparisons offer suggestions for the design of specific drugs. Furthermore, the new structure represents an intermediate conformation between the open apo form and the closed holo form observed previously, giving insights into the conformational changes associated with catalysis.

  • 22.
    Henriksson, Lena M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Björkelid, Christofer
    Mowbray, Sherry L.
    Unge, Torsten
    The 1.9 Å resolution structure of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase, a potential drug target2006In: Acta Crystallogr. Section D Biol. Crystallogr., ISSN 0907-4449, Vol. 62, p. 807-813Article in journal (Refereed)
  • 23.
    Henriksson, Lena M
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Structural Biology.
    Johansson, Patrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Structural Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Structural Biology.
    Mowbray, Sherry L
    X-ray structure of peptidyl-prolyl cis-trans isomerase A from Mycobacterium tuberculosis.2004In: Eur J Biochem, ISSN 0014-2956, Vol. 271, no 20, p. 4107-13Article in journal (Refereed)
  • 24.
    Henriksson, Lena M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mowbray, Sherry L.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 27, p. 19905-19916Article in journal (Refereed)
    Abstract [en]

    Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn2+, NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC50 value of 80 nM. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.

  • 25. Hsiao, An-Shan
    et al.
    Haslam, Richard P.
    Michaelson, Louise V.
    Liao, Pan
    Chen, Qin-Fang
    Sooriyaarachchi, Sanjeewani
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Napier, Johnathan A.
    Tanner, Julian A.
    Chye, Mee-Len
    Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development2014In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 34, no 6, p. 865-877Article in journal (Refereed)
    Abstract [en]

    Eukaryotic cytosolic ACBPs (acyl-CoA-binding proteins) bind acyl-CoA esters and maintain a cytosolic acyl-CoA pool, but the thermodynamics of their protein-lipid interactions and physiological relevance in plants are not well understood. Arabidopsis has three cytosolic ACBPs which have been identified as AtACBP4, AtACBP5 and AtACBP6, and microarray data indicated that all of them are expressed in seeds; AtACBP4 is expressed in early embryogenesis, whereas AtACBP5 is expressed later. ITC (isothermal titration calorimetry) in combination with transgenic Arabidopsis lines were used to investigate the roles of these three ACBPs from Arabidopsis thaliana. The dissociation constants, stoichiometry and enthalpy change of AtACBP interactions with various acyl-CoA esters were determined using ITC. Strong binding of recombinant (r) AtACBP6 with long-chain acyl-CoA (C16-to C18-CoA) esters was observed with dissociation constants in the nanomolar range. However, the affinity of rAtACBP4 and rAtACBP5 to these acyl-CoA esters was much weaker (dissociation constants in the micromolar range), suggesting that they interact with acyl-CoA esters differently from rAtACBP6. When transgenic Arabidopsis expressing AtACBP6pro::GUS was generated, strong GUS (beta-glucuronidase) expression in cotyledonary-staged embryos and seedlings prompted us to measure the acyl-CoA contents of the acbp6 mutant. This mutant accumulated higher levels of C18:1-CoA and C18:1- and C18:2-CoAs in cotyledonary-staged embryos and seedlings, respectively, in comparison with the wild type. The acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to abscisic acid during germination, suggesting their physiological functions in seeds.

  • 26.
    Jansson, Anna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Więckowska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Yahiaoui, Samir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Sooriyaarachchi, Sanjeewani
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Lindh, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dharavath, Shyamraj
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Desroses, Matthieu
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Suresh, Surisetti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Andaloussi, Mounir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Nikhil, Rautela
    AstraZeneca India Private Limited.
    Sreevalli, Sharma
    AstraZeneca India Private Limited.
    Srinivasa, Bachally R.
    AstraZeneca India Private Limited.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    DXR Inhibition by Potent Mono- and Disubstituted Fosmidomycin Analogues2013In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 56, no 15, p. 6190-6199Article in journal (Refereed)
    Abstract [en]

    The antimalarial compound fosmidomycin targets DXR, the enzyme that catalyzes the first committed step in the MEP pathway producing the universally essential isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The MEP pathway is used by a number of pathogens, including Mycobacterium tuberculosis and apicomplexan parasites, and differs from the classical mevalonate pathway that is essential in humans. Using a structure-based approach, we designed a number of analogues of fosmidomycin, including a series that are substituted in both the Cα and the hydroxamate positions. The latter proved to be a stable framework for the design of inhibitors that extend from the cramped substrate-binding site and can, for the first time, bridge the substrate and cofactor binding sites. A number of these compounds are more potent than fosmidomycin in terms of killing Plasmodium falciparum in an in vitro assay; the best has an IC50 of 40 nM.

  • 27.
    Johansson, Patrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Cronin, Annette
    Arand, Michael
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Jones, T Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Structural Biology Program.
    Mowbray, Sherry L
    Structure of an atypical epoxide hydrolase from Mycobacterium tuberculosis2005In: J Mol Biol, ISSN 0022-2836, Vol. 351, no 5, p. 1048-56Article in journal (Refereed)
  • 28.
    Krajewski, Wojciech W.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Collins, Ruairi
    Holmberg-Schiavone, Lovisa
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Karlberg, Tobias
    Mowbray, Sherry L.
    Crystal Structures of Mammalian Glutamine Synthetases Illustrate Substrate-Induced Conformational Changes and Provide Opportunities for Drug and Herbicide Design2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 375, no 1, p. 217-228Article in journal (Refereed)
    Abstract [en]

    Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 Å. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 Å and 2.6 Å, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.

  • 29.
    Krajewski, Wojciech W.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Jones, T. Alwyn
    Mowbray, Sherry L.
    Structure of Mycobacterium tuberculosis glutamine synthetase in complex with a transition-state mimic provides functional insights2005In: Proc. Natl. Acad. Sci. USA, ISSN 0027-8424, Vol. 102, no 30, p. 10499-10504Article in journal (Refereed)
  • 30.
    Krajewski, Wojciech W
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Jones, T Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mowbray, Sherry L
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Structure of Mycobacterium tuberculosis glutamine synthetase in complex with a transition-state mimic provides functional insights2005In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 102, no 30, p. 10499-10504Article in journal (Refereed)
    Abstract [en]

    Glutamine synthetase catalyzes the ligation of glutamate and ammonia to form glutamine, with the resulting hydrolysis of ATP. The enzyme is a central component of bacterial nitrogen metabolism and is a potential drug target. Here, we report a high-yield recombinant expression system for glutamine synthetase of Mycobacterium tuberculosis together with a simple purification. The procedure allowed the structure of a complex with a phosphorylated form of the inhibitor methionine sulfoximine, magnesium, and ADP to be solved by molecular replacement and refined at 2.1-angstrom resolution. To our knowledge, this study provides the first reported structure for a taut form of the M. tuberculosis enzyme, similar to that observed for the Salmonella enzyme earlier. The phospho compound, generated in situ by an active enzyme, mimics the phosphorylated tetrahedral adduct at the transition state. Some differences in ligand interactions of the protein with both phosphorylated compound and nucleotide are observed compared with earlier structures; a third metal ion also is found. The importance of these differences in the catalytic mechanism is discussed; the results will help guide the search for specific inhibitors of potential therapeutic interest.

  • 31.
    Lagerlund, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Odell, Luke R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Mowbray, Sherry L.
    Nilsson, Mikael T.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Krajewski, Wojciech
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Nordqvist, Anneli
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Microwave-enhanced alpha-arylation of a protected glycine in water: evaluation of 3-phenylglycine derivatives as inhibitors of the tuberculosis enzyme, glutamine synthetase2007In: Combinatorial chemistry & high throughput screening, ISSN 1386-2073, E-ISSN 1875-5402, Vol. 10, no 9, p. 783-789Article in journal (Refereed)
    Abstract [en]

    A microwave-enhanced, palladium-catalyzed protocol for the alpha-arylation of a protected glycine in neat water is described. This reaction proceeds rapidly, under non-inert conditions, to afford a range of phenylglycine derivatives in moderate to good yields. Based on this arylation, a number of aryl L-methionine-SR-sulfoximine (MSO) analogues were prepared and evaluated for their Mycobacterium tuberculosis glutamine synthetase (TB-GS) inhibitory activity.

  • 32.
    Magnusson, Ulrika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Chaudhuri, Barnali Neel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ko, Junsang
    Park, Chankyu
    Jones, T Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mowbray, Sherry L
    Hinge-bending motion of D-allose-binding protein from Escherichia coli2002In: J Biol Chem, ISSN 0021-9258, Vol. 277, no 16, p. 14077-84Article in journal (Refereed)
  • 33.
    Magnusson, Ulrika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Luck, Linda A.
    Mowbray, Sherry L.
    X-ray structures of the leucine-binding protein illustrate conformational changes, as well as ligand specificityManuscript (Other academic)
  • 34.
    Magnusson, Ulrika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Structural Biology.
    Salopek-Sondi, Branka
    Luck, Linda A
    Mowbray, Sherry L
    X-ray structures of the leucine-binding protein illustrate conformational changes and the basis of ligand specificity.2004In: J Biol Chem, ISSN 0021-9258, Vol. 279, no 10, p. 8747-52Article in journal (Refereed)
  • 35. Mariano, Sandrine
    et al.
    Roos, Annette K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mowbray, Sherry L.
    Salmon, Laurent
    Competitive inhibitors of type B ribose 5-phosphate isomerases: design, synthesis and kinetic evaluation of new D-allose and D-allulose 6-phosphate derivatives2009In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 344, no 7, p. 869-880Article in journal (Refereed)
    Abstract [en]

    This study reports syntheses Of D-allose 6-phosphate (All6P), D-allulose (or D-psicose) 6-phosphate (Allu6P), and seven D-ribose 5-phosphate isomerase (Rpi) inhibitors. The inhibitors were designed as analogues of the 6-carbon high-energy intermediate postulated for the All6P to Allu6P isomerization reaction (Allpi activity) catalyzed by type B Rpi from Escherichia coli (EcRpiB). 5-PhosphO-D-ribonate, easily obtained through oxidative cleavage of either All6P or Allu6P, led to the original synthon 5-dihydrogenopliospho-D-ribono-1,4-lactone from which the other inhibitors could be synthesized through nucleophilic addition in one step. Kinetic evaluation on Allpi activity of EcRpiB shows that two of these compounds. 5phospho-D-ribonohydroxamic acid and N-(5-phospho-D-ribonoyl)-methylamine, indeed behave as new efficient inhibitors of EcRpiB; further, 5-phospho-D-ribonohydroxamic acid was demonstrated to have competitive inhibition. Kinetic evaluation on Rpi activity of both EcRpiB and RpiB from Mycobacterium tuberculosis (MtRpiB) shows that several of the designed 6-carbon high-energy intermediate analogues are new competitive inhibitors of both RpiBs. One of them, 5-phospho-D-ribonate, not only appears as the strongest competitive inhibitor of a Rpi ever reported in the literature, with a K-i value of 9 mu M for MtRpiB, but also displays specific inhibition of MtRpiB versus EcRpiB.

  • 36.
    Mowbray, Sherry L.
    et al.
    Swedish University of Agricultural Sciences, Department of Molecular Biology.
    Elfström, Lisa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Ahlgren, Kerstin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andersson, Evalena
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    X-ray structure of potato epoxide hydrolase sheds light on its substrate specificity2006In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 15, no 7, p. 1628-1637Article in journal (Refereed)
    Abstract [en]

    Abstract: Epoxide hydrolases catalyze the conversion of epoxides to diols. The known functions of such enzymes include detoxification of xenobiotics, drug metabolism, synthesis of signaling compounds, and intermediary metabolism. In plants, epoxide hydrolases are thought to participate in general defense systems. In the present study, we report the first structure of a plant epoxide hydrolase, one of the four homologous enzymes found in potato. The structure was solved by molecular replacement and refined to a resolution of 1.95 angstrom. Analysis of the structure allows a better understanding of the observed substrate specificities and activity. Further, comparisons with mammalian and fungal epoxide hydrolase structures reported earlier show the basis of differing substrate specificities in the various epoxide hydrolase subfamilies. Most plant enzymes, like the potato epoxide hydrolase, are expected to be monomers with a preference for substrates with long lipid-like substituents of the epoxide ring. The significance of these results in the context of biological roles and industrial applications is discussed.

  • 37.
    Mowbray, Sherry L
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kathiravan, Muthu K
    Pandey, Abhishek A
    Odell, Luke R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Inhibition of Glutamine Synthetase: A Potential Drug Target in Mycobacterium tuberculosis2014In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 19, no 9, p. 13161-13176Article, review/survey (Refereed)
    Abstract [en]

    Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Globally, tuberculosis is second only to AIDS in mortality and the disease is responsible for over 1.3 million deaths each year. The impractically long treatment schedules (generally 6-9 months) and unpleasant side effects of the current drugs often lead to poor patient compliance, which in turn has resulted in the emergence of multi-, extensively- and totally-drug resistant strains. The development of new classes of anti-tuberculosis drugs and new drug targets is of global importance, since attacking the bacterium using multiple strategies provides the best means to prevent resistance. This review presents an overview of the various strategies and compounds utilized to inhibit glutamine synthetase, a promising target for the development of drugs for TB therapy.

  • 38.
    Mowbray, SL
    et al.
    Uppsala University.
    Bjorkman, AJ
    Uppsala University.
    Conformational changes of ribose-binding protein and two related repressors are tailored to fit the functional need1999In: JOURNAL OF MOLECULAR BIOLOGY, ISSN 0022-2836, Vol. 294, no 2, p. 487-499Article in journal (Other academic)
    Abstract [en]

    The structures and conformational changes of the periplasmic ribose-binding protein and two repressors, PurR and LacI, were compared. Although the closed, ligand-bound structures of the three proteins are very similar, they differ greatly in the degree an

  • 39.
    Mowbray, SL
    et al.
    Uppsala University.
    Helgstrand, C
    Uppsala University.
    Sigrell, JA
    Uppsala University.
    Cameron, AD
    Uppsala University.
    Jones, TA
    Uppsala University.
    Errors and reproducibility in electron-density map interpretation1999In: ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, ISSN 0907-4449, Vol. 55, p. 1309-1319Article in journal (Other academic)
    Abstract [en]

    Three investigators, with varying levels of experience, independently built and refined the structure of Escherichia coli ribokinase at 2.6 Angstrom resolution. At the end of the refinement/rebuilding processes the models had essentially converged, althou

  • 40. Mowbray, SL
    et al.
    Sandgren, MOJ
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Chemotaxis receptors: A progress report on structure and function1998In: JOURNAL OF STRUCTURAL BIOLOGY, Vol. 124, no 2-3, p. 257-275Article, book review (Other (popular science, discussion, etc.))
    Abstract [en]

    Recent biochemical and structural studies have provided many new insights into the structure and function of bacterial chemoreceptors. Aspects of their ligand binding, conformational changes, and interactions with other members of the signaling pathway ar

  • 41.
    Nilsson, Mikael T.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Krajewski, Wojciech W.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Srinivasa, Bachally R.
    Yahiaoui, Samir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mowbray, Sherry L.
    Structural basis for the inhibition of Mycobacterium tuberculosis glutamine synthetase by novel ATP-competitive inhibitors2009In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 393, no 2, p. 504-513Article in journal (Refereed)
    Abstract [en]

    Glutamine synthetase (GS, EC 6.3.1.2; also known as γ-glutamyl:ammonia ligase) catalyzes the ATP-dependent condensation of glutamate and ammonia to form glutamine. The enzyme has essential roles in different tissues and species, which have led to its consideration as a drug or an herbicide target. In this article, we describe studies aimed at the discovery of new antimicrobial agents targeting Mycobacterium tuberculosis, the causative pathogen of tuberculosis. A number of distinct classes of GS inhibitors with an IC50 of micromolar value or better were identified via high-throughput screening. A commercially available purine analogue similar to one of the clusters identified (the diketopurines), 1-[(3,4-dichlorophenyl)methyl]-3,7-dimethyl-8-morpholin-4-yl-purine-2,6-dione, was also shown to inhibit the enzyme, with a measured IC50 of 2.5 ± 0.4 μM. Two X-ray structures are presented: one is a complex of the enzyme with the purine analogue alone (2.55-Å resolution), and the other includes the compound together with methionine sulfoximine phosphate, magnesium and phosphate (2.2-Å resolution). The former represents a relaxed, inactive conformation of the enzyme, while the latter is a taut, active one. These structures show that the compound binds at the same position in the nucleotide site, regardless of the conformational state. The ATP-binding site of the human enzyme differs substantially, explaining why it has an ∼ 60-fold lower affinity for this compound than the bacterial GS. As part of this work, we devised a new synthetic procedure for generating l-(SR)-methionine sulfoximine phosphate from l-(SR)-methionine sulfoximine, which will facilitate future investigations of novel GS inhibitors.

  • 42.
    Nordqvist, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Andaloussi, Mounir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Jansson, Anna M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Synthesis of Functionalized Cinnamaldehyde Derivatives by an Oxidative Heck Reaction and Their Use as Starting Materials for Preparation of Mycobacterium tuberculosis 1-Deoxy-D-xylulose-5-phosphate Reductoisomerase Inhibitors2011In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 76, no 21, p. 8986-8998Article in journal (Refereed)
    Abstract [en]

    Cinnamaldehyde derivatives were synthesized in good to excellent yields in one step by a mild and selective, base-free palladium(II)-catalyzed oxidative Heck reaction starting from acrolein and various arylboronic acids. Prepared α,β-unsaturated aldehydes were used for synthesis of novel α-aryl substituted fosmidomycin analogues, which were evaluated for their inhibition of Mycobacterium tuberculosis 1-deoxy-d-xylulose 5-phosphate reductoisomerase. IC(50) values between 0.8 and 27.3 μM were measured. The best compound showed activity comparable to that of the most potent previously reported α-aryl substituted fosmidomycin-class inhibitor.

  • 43.
    Nordqvist, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Nilsson, Mikael T.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Lagerlund, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Muthas, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Yahiaoui, Samir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Odell, Luke R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Srinivasa, Bachally R.
    Astra Research Center India, Bangalore, India.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Synthesis, biological evaluation and X-ray crystallographic studies of imidazo[1,2-a]-pyridine based Mycobacterium tuberculosis glutamine synthetase inhibitors2012In: MedChemComm, ISSN 2040-2503, E-ISSN 2040-2511, Vol. 3, no 5, p. 620-626Article in journal (Refereed)
    Abstract [en]

    Based on an imidazo[1,2-a]pyridine hit from a high-throughput screen directed at the M. tuberculosis enzyme glutamine synthetase, a hit expansion was performed by synthesizing a series of analogs. A set of 16 molecules was first synthesized according to a statistical molecular design approach. One potent inhibitor was identified (IC50 = 3.0 µM), which led to the synthesis of 17 additional imidazo[1,2-a]pyridines in a follow-up study. Among these, several inhibitors were identified showing single digit micromolar potency. An X-ray structure of one of these revealed the binding mode of this class of inhibitors in the ATP-binding site, and allowed us to rationalize some of the structure-activity relationships observed.

  • 44.
    Nordqvist, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Nilsson, Mikael T.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Röttger, Svenja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Odell, Luke R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Krajewski, Wojciech W.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Andersson, C. Evalena
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery2008In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 16, no 10, p. 5501-5513Article in journal (Refereed)
    Abstract [en]

    A combination of a literature survey, structure-based virtual screening and synthesis of a small library was performed to identify hits to the potential antimycobacterial drug target, glutamine synthetase. The best inhibitor identified from the literature survey was (2S,5R)-2,6-diamino-5-hydroxyhexanoic acid (4, IC(50) of 610+/-15microM). In the virtual screening 46,400 compounds were docked and subjected to a pharmacophore search. Of these compounds, 29 were purchased and tested in a biological assay, allowing three novel inhibitors containing an aromatic scaffold to be identified. Based on one of the hits from the virtual screening a small library of 15 analogues was synthesized producing four compounds that inhibited glutamine synthetase.

  • 45.
    Persson, Camilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Mowbray, Sherry
    Östman, Arne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Primary sequence determinants responsible for site-selective dephosphorylation of the PDGF beta-receptor by the receptor-like protein tyrosine phosphatase DEP-12002In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 517, no 1-3, p. 27-31Article in journal (Refereed)
    Abstract [en]

    Site-selective dephosphorylation of receptor tyrosine kinases contributes to receptor regulation. The receptor-like protein tyrosine phosphatase DEP-1 site-selectively dephosphorylates the PDGF beta-receptor. DEP-1 dephosphorylation of original and chimeric phospho-peptides spanning the preferred pY1021 and the less preferred pY857 and pY562 sites was analyzed. Double substitutions of basic residues at -4 and +3 of pY857 and pY562 peptides improved affinity. Substitutions of single amino acids indicated preference for an acidic residue at position -1 and a preference against a basic residue at position +3. DEP-1 site-selective dephosphorylation of PDGF beta-receptor is thus determined by the primary sequence surrounding phosphorylation sites and involves interactions with residues spanning at least between positions -1 and +3.

  • 46. Reetz, Manfred T.
    et al.
    Bocola, Marco
    Wang, Li-Wen
    Sanchis, Joaquin
    Cronin, Annette
    Arand, Michael
    Zou, Jinyu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Archelas, Alain
    Bottalla, Anne-Lise
    Naworyta, Agata
    Mowbray, Sherry L.
    Directed Evolution of an Enantioselective Epoxide Hydrolase: Uncovering the Source of Enantioselectivity at Each Evolutionary Stage2009In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 21, p. 7334-7343Article, review/survey (Refereed)
    Abstract [en]

    Directed evolution of enzymes as enantioselective catalysts in organic chemistry is an alternative to traditional asymmetric catalysis using chiral transition-metal complexes or organocatalysts, the different approaches often being complementary. Moreover, directed evolution studies allow us to learn more about how enzymes perform mechanistically. The present study concerns a previously evolved highly enantioselective mutant of the epoxide hydrolase from Aspergillus niger in the hydrolytic kinetic resolution of racemic glycidyl phenyl ether. Kinetic data, molecular dynamics calculations, molecular modeling, inhibition experiments, and X-ray structural work for the wild-type (WT) enzyme and the best mutant reveal the basis of the large increase in enantioselectivity (E = 4.6 versus E = 115). The overall structures of the WT and the mutant are essentially identical, but dramatic differences are observed in the active site as revealed by the X-ray structures. All of the experimental and computational results support a model in which productive positioning of the preferred (S)-glycidyl phenyl ether, but not the (R)-enantiomer, forms the basis of enhanced enantioselectivity. Predictions regarding substrate scope and enantioselectivity of the best mutant are shown to be possible.

  • 47. Roos, Annette
    et al.
    Andersson, Evalena
    Bergfors, Terese
    Jacobsson, Micael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Karlén, Anders
    Unge, Torsten
    Jones, Alwyn
    Mowbray, Sherry
    Mycobacterium tuberculosis ribose-5-phosphate isomerase has a known fold, but a novel active site2004In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 335, no 3, p. 799-809Article in journal (Refereed)
  • 48.
    Roos, Annette K
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Structural Biology.
    Andersson, C Evalena
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Structural Biology.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Structural Biology.
    Jacobsson, Micael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Structural Biology.
    Jones, T Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Structural Biology.
    Mowbray, Sherry L
    Mycobacterium tuberculosis ribose-5-phosphate isomerase has a known fold, but a novel active site.2004In: J Mol Biol, ISSN 0022-2836, Vol. 335, no 3, p. 799-809Article in journal (Refereed)
  • 49.
    Roos, Annette K
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Structural Biology Program.
    Burgos, Emmanuel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Ericsson, Daniel J
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Salmon, Laurent
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mowbray, Sherry L
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Competitive inhibitors of Mycobacterium tuberculosis ribose-5-phosphate isomerase2005In: J Biol Chem, ISSN 0021-9258, Vol. 280, no 8, p. 6416-22Article in journal (Refereed)
  • 50.
    Roos, Annette K.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mariano, Sandrine
    Kowalinski, Eva
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Salmon, Laurent
    Mowbray, Sherry L.
    D-ribose-5-phosphate isomerase B from Escherichia coli is also a functional D-allose-6-phosphate isomerase, while the Mycobacterium tuberculosis enzyme is not2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 382, no 3, p. 667-679Article in journal (Refereed)
    Abstract [en]

    Interconversion of D-ribose-5-phosphate (R5P) and D-ribulose-5-phosphate is an important step in the pentose phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars D-allose-6-phosphate (All6P) and D-allulose-6-phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme's primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-D-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme's structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate ribose-5-phosphate, shed additional light on the reaction mechanism of RpiBs generally.

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