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  • 1. Abdurahman, Samir
    et al.
    Höglund, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Höglund, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Vahlne, Anders
    Mutation in the loop C-terminal to the cyclophilin A binding site of HIV-1 capsid protein disrupts proper virus assembly and infectivity2007In: Retrovirology, ISSN 1742-4690, E-ISSN 1742-4690, Vol. 4, p. 19-Article in journal (Refereed)
    Abstract [en]

    We have studied the effects associated with two single amino acid substitution mutations in HIV-1 capsid (CA), the E98A and E187G. Both amino acids are well conserved among all major HIV-1 subtypes. HIV-1 infectivity is critically dependent on proper CA cone formation and mutations in CA are lethal when they inhibit CA assembly by destabilizing the intra and/or inter molecular CA contacts, which ultimately abrogate viral replication. Glu98, which is located on a surface of a flexible cyclophilin A binding loop is not involved in any intra-molecular contacts with other CA residues. In contrast, Glu187 has extensive intra-molecular contacts with eight other CA residues. Additionally, Glu187 has been shown to form a salt-bridge with Arg18 of another N-terminal CA monomer in a N-C dimer. However, despite proper virus release, glycoprotein incorporation and Gag processing, electron microscopy analysis revealed that, in contrast to the E187G mutant, only the E98A particles had aberrant core morphology that resulted in loss of infectivity.

  • 2. Akoachere, Monique
    et al.
    Iozef, Rimma
    Rahlfs, Stefan
    Deponte, Marcel
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Creighton, Donald J
    Schirmer, Heiner
    Becker, Katja
    Characterization of the glyoxalases of the malarial parasite Plasmodium falciparum and comparison with their human counterparts.2005In: Biol Chem, ISSN 1431-6730, Vol. 386, no 1, p. 41-52Article in journal (Refereed)
    Abstract [en]

    The glyoxalase system consisting of glyoxalase I (GloI) and glyoxalase II (GloII) constitutes a glutathione-dependent intracellular pathway converting toxic 2-oxoaldehydes, such as methylglyoxal, to the corresponding 2-hydroxyacids. Here we describe a complete glyoxalase system in the malarial parasite Plasmodium falciparum. The biochemical, kinetic and structural properties of cytosolic GloI (cGloI) and two GloIIs (cytosolic GloII named cGloII, and tGloII preceded by a targeting sequence) were directly compared with the respective isofunctional host enzymes. cGloI and cGloII exhibit lower K(m) values and higher catalytic efficiencies (k(cat)/K(m) ) than the human counterparts, pointing to the importance of the system in malarial parasites. A Tyr185Phe mutant of cGloII shows a 2.5-fold increase in K(m) , proving the contribution of Tyr185 to substrate binding. Molecular models suggest very similar active sites/metal binding sites of parasite and host cell enzymes. However, a fourth protein, which has highest similarities to GloI, was found to be unique for malarial parasites; it is likely to act in the apicoplast, and has as yet undefined substrate specificity. Various S-(N-hydroxy-N-arylcarbamoyl)glutathiones tested as P. falciparum Glo inhibitors were active in the lower nanomolar range. The Glo system of Plasmodium will be further evaluated as a target for the development of antimalarial drugs.

  • 3.
    Andersson, Malena
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Exploring protein evolution by saturation mutagenesis of the GST M2-2 active site residue 2102005In: FEBS Journal, Vol. 272, p. 81 Suppl-Article in journal (Other scientific)
  • 4. Bacskay, Ivett
    et al.
    Takatsy, Aniko
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Vegvari, Akos
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Elfwing, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology, Centre for Surface Biotechnology.
    Balllagi-Pordany, Andras
    Kilar, Ferenc
    Hjertén, Stellan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses, and cells (bacteria). III: Gel antibodies against cells (bacteria)2006In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 27, no 23, p. 4682-4687Article in journal (Refereed)
    Abstract [en]

    Artificial antibodies in the form of gel granules were synthesized from the monomers acrylamide and N,N'-methylenebisacrylamide by the imprinting method in the presence of Echerichia coli bacteria as template. The electrophoretic migration velocities of the gel antibodies (i) saturated with the antigen (Escherichia coli MRE-600), (ii) freed of the antigen, and (iii) resaturated with bacteria, were determinated by electrophoresis in a rotating narrow-bore tube of 245 mm length and the 2.5 and 9.6 mm inner and outer diameters, respectively. Removal of bacteria from the gel antibodies was made by treatment with enzymes, followed by washing with SDS and buffer. Gel granules becoming charged by adsorption of bacteria move in an electrical field. We obtained a significant selectivity of gel antibodies for E coli MRE-600, since the granules did not interact with Lactococcus lactis; and when E coli BL21 bacteria were added to the gels selective for E coli MRE-600, a significant difference in the migration rate of the complexes formed with the two strains was observed indicating the ability of differentiation between the two strains. The gel antibodies can be used repeatedly. The new imprinting method for the synthesis of artificial gel antibodies against bioparticles described herein, and the classical electrophoretic analysis technique employed, thus represent - when combined - a new approach to distinguish between different types and strains of bacteria. The application area can certainly be extended to cover other classes of cells.

  • 5.
    Barman, Jharna
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Acharya, Sandipta
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Zhou, Chuanzheng
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Chatterjee, Subhrangsu
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Engström, Åke
    Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Non-identical electronic characters of the internucleotidic phosphates in RNA modulate the chemical reactivity of the phosphodiester bonds.2006In: Org Biomol Chem, ISSN 1477-0520, Vol. 4, no 5, p. 928-41Article in journal (Refereed)
  • 6. Beyer, B
    et al.
    Rubin, J
    Johnson, J
    Chung, A
    Hayashi, Y
    Andersson, D
    Kiso, Y
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Dunn, B
    Candida albicans secreted aspartic peptidase specificity: A chromogenic combinatorial approach2003In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 71, no 3, p. 310-P025Article in journal (Other academic)
  • 7. Bodin, Karl
    et al.
    Andersson, Ulla
    Rystedt, Eva
    Ellis, Eva
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Pikuleva, Irina
    Eggertsen, Gösta
    Björkhem, Ingemar
    Diczfalusy, Ulf
    Metabolism of 4 beta -hydroxycholesterol in humans2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 35, p. 31534-31540Article in journal (Refereed)
    Abstract [en]

    One of the major oxysterols in the human circulation is 4 beta-hydroxycholesterol formed from cholesterol by the drug-metabolizing enzyme cytochrome P450 3A4. Deuterium-labeled 4 beta-hydroxycholesterol was injected into two healthy volunteers, and the apparent half-life was found to be 64 and 60 h, respectively. We have determined earlier the half-lives for 7 alpha-, 27-, and 24-hydroxycholesterol to be approximately 0.5, 0.75, and 14 h, respectively. Patients treated with certain antiepileptic drugs have up to 20-fold increased plasma concentrations of 4 beta-hydroxycholesterol. The apparent half-life of deuterium-labeled 4 beta-hydroxycholesterol in such a patient was found to be 52 h, suggesting that the high plasma concentration was because of increased synthesis rather than impaired clearance. 4 beta-Hydroxycholesterol was converted into acidic products at a much slower rate than 7 alpha-hydroxycholesterol in primary human hepatocytes, and 4 beta-hydroxycholesterol was 7 alpha-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4 beta-hydroxycholesterol. These results suggest that the high plasma concentration of 4 beta-hydroxycholesterol is because of its exceptionally slow elimination, probably in part because of the low rate of 7 alpha-hydroxylation of the steroid. The findings are discussed in relation to a potential role of 4 beta-hydroxycholesterol as a ligand for the nuclear receptor LXR.

  • 8. Brandt, Peter
    et al.
    Geitmann, Matthis
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Deconstruction of Non-Nucleoside Reverse Transcriptase Inhibitors of Human Immunodeficiency Virus Type 1 for Exploration of the Optimization Landscape of Fragments2011In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804Article in journal (Refereed)
    Abstract [en]

    This study has taken a closer look at the theoretical basis for protein-fragment interactions. The approach involved the deconstruction of 3 non-nucleoside inhibitors of HIV-1 reverse transcriptase and investigation of the interaction between 21 substructures and the enzyme. It focused on the concept of ligand efficiency and showed that ligand independent free energy fees (ΔG(ind)) are crucial for the understanding of the binding affinities of fragments. A value of 7.0 kcal mol(-1) for the ΔG(ind) term is shown to be a lower limit for the NNRTI binding pocket of HIV-1 RT. The addition of the ΔG(ind) term to the dissociation free energy in the calculation of a corrected ligand efficiency, in combination with the lack of an efficient ligand binding hot spot in the NNIBP, fully explains the existence of nonbinding NNRTI substructures. By applying the concept to a larger set of ligands, we could define a binding site profile that indicates the absence of an efficient fragment binding hot spot but an efficient binding of full-sized NNRTIs. The analysis explains some of the challenges in identifying fragments against flexible targets involving conformational changes and how fragments may be prioritized.

  • 9. Brunnström, Åsa
    et al.
    Hamberg, Mats
    Griffiths, William J.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Claesson, Hans-Erik
    Biosynthesis of 14,15-Hepoxilins in Human L1236 Hodgkin Lymphoma Cells and Eosinophils2011In: Lipids, ISSN 0024-4201, E-ISSN 1558-9307, Vol. 46, no 1, p. 69-79Article in journal (Refereed)
    Abstract [en]

    Hepoxilins are epoxy alcohols synthesized through the 12-lipoxygenase (12-LO) pathway in animal cells. The epidermis is the principal source of hepoxilins in humans. Here we report on the formation of novel hepoxilin regioisomers formed by the 15-LO pathway in human cells. The Hodgkin lymphoma cell line L1236 possesses high 15-lipoxygenase-1 (15-LO-1) activity and incubation of L1236 cells with arachidonic acid led to the formation of 11(S)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),12(E) eicosatrienoic acid (14,15-HxA(3) 11(S)) and 13(R)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),11(Z) eicosatrienoic acid (14,15-HxB(3) 13 (R)). In addition, two hitherto unidentified products were detected and these products were collected and analyzed by positive ion electrospray tandem mass spectrometry. These metabolites were identified as 11(S),15(S)-dihydroxy-14(R)-glutathionyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA(3)-C) and 11(S),15(S)-dihydroxy-14(R)-cysteinyl-glycyl-5(Z),8(Z),12(E)-eicosatrien oic acid (14,15-HxA(3)-D). Incubation of L1236 cells with synthetic 14,15-HxA(3) 11(S) also led to the formation of 14,15-HxA(3)-C and 14,15-HxA(3)-D. Several soluble glutathione transferases, in particular GST M1-1 and GST P1-1, were found to catalyze the conversion of 14,15-HxA(3) to 14,15-HxA(3)-C. L1236 cells produced approximately twice as much eoxins as cysteinyl-containing hepoxilins upon stimulation with arachidonic acid. Human eosinophils, nasal polyps and dendritic cells selectively formed 14,15-HxA(3) 11(S) and 14,15-HxB(3) 13(R) stereoisomers, but not cysteinyl-containing hepoxilins, after stimulation with arachidonic acid. Furthermore, purified recombinant 15-LO-1 alone catalyzed the conversion of arachidonic acid to 14,15-HxA(3) 11(S) and 14,15-HxB(3) 13(R), showing that human 15-LO-1 possesses intrinsic 14,15-hepoxilin synthase activity.

  • 10. Cassimjee, Karim Engelmark
    et al.
    Kourist, Robert
    Lindberg, Diana
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Wittrup Larsen, Marianne
    Thanh, Nguyen Hong
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Bornscheuer, Uwe T
    Berglund, Per
    One-step enzyme extraction and immobilization for biocatalysis applications2011In: Biotechnology Journal, ISSN 1860-6768, Vol. 6, no 4, p. 463-469Article in journal (Refereed)
    Abstract [en]

    An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.

  • 11.
    Chaga, Grigoriy
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Andersson, Lennart
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Porath, Jerker
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes1994In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 7, no 9, p. 1115-1119Article in journal (Refereed)
    Abstract [en]

    Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid residues, His84 and His85, that are suitably positioned for coordination to Ni(II)-IDA-agarose. This particular structural motif is lacking in GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineering afforded a mutant enzyme that displays affinity for Ni(II)-IDA-agarose, in contrast to the wild-type GST M1-1. The results identify a distinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins.

  • 12.
    Christopeit, Tony
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Stenberg, Gun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gossas, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Nyström, Susanne
    Baraznenok, Vera
    Lindström, Erik
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 414, no 1, p. 14-22Article in journal (Refereed)
    Abstract [en]

    A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (β-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer's disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer's disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.

  • 13.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Fragment library screening and lead characterization using SPR biosensors2009In: Current Topics in Medicinal Chemistry, ISSN 1568-0266, E-ISSN 1873-4294, Vol. 9, no 18, p. 1725-1735Article in journal (Refereed)
    Abstract [en]

    The transition from high throughput screening of collections of drug-like compounds to screening of fragment libraries via lower throughput methods with high sensitivity has revolutionized early drug discovery. It is highlighting the need for sensitive biophysical techniques for interaction analysis rather than high throughput methods. Biosensors with SPR detection are well suited for this novel scenario. In less than 20 years the technique has been launched, established and become a highly informative method for a variety of applications in drug discovery. It is no longer limited to the detection of proteins or other high molecular weight analytes, but the detection of weakly interacting fragments is now feasible. This paper discusses the theoretical and experimental limitations for such applications and reviews a number of successful studies in the area of fragment-based lead discovery that have recently been published. It can be anticipated that the evolution of this young technique will be significantly influenced by the requirements for efficient fragment-based lead discovery.

  • 14.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Esterbauer, Hermann
    Mannervik, Bengt
    Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases1987In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 247, no 3, p. 707-713Article in journal (Refereed)
    Abstract [en]

    The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.

  • 15.
    Danielson, U. Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Jiang, Fanyi Y
    Hansson, Lars O.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Probing the kinetic mechanism and coenzyme specificity of glutathione reductase from the cyanobacterium Anabaena PCC 7120 by redesign of the pyridine-nucleotide-binding site1999In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 38, no 29, p. 9254-9263Article in journal (Refereed)
    Abstract [en]

    Glutathione reductase from the cyanobacterium Anabaena PCC 7120 contains a pyridine-nucleotide-binding motif differing from that of the enzyme from other sources and an insertion of 10 amino acid residues. Homology modeling was used to obtain a model of the enzyme structure. It revealed that in the Anabaena enzyme Lys(203) replaces Arg, found to interact with the 2'-phosphate of NADP(H) in the enzyme from other sources, and that it has an extra loop near the entrance of the pyridine-nucleotide-binding site. The steady-state and preequilibrium kinetic properties were characterized for the wild-type enzyme, a K203R, and a loop deletion mutant. All enzyme forms had higher catalytic efficiency with NADPH than with NADH, although the difference was less than for glutathione reductase from other sources. The specificity was most pronounced in the formation of the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD, as compared to later steps in the reaction. Unexpectedly, by replacing Lys(203) with Arg, the specificity for NADPH was diminished in the complete redox reaction. Ser(174) appears to interact with the 2'-phosphate of NADPH and introduction of arginine instead of lysine, therefore, has little effect on the interaction with this coenzyme. However, the efficiency in forming the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD was increased in the K203R mutant using NADPH but not with NADH. The lack of affinity toward 2',5'-ADP-Sepharose by the wild-type enzyme was not changed by replacing Lys(203) with Arg but deletion of the loop resulted in an enzyme that bound to the immobilized ligand. Removal of the loop increased the efficiency of the enzyme in the reductive half-reaction with both pyridine-nucleotides as well as in the overall catalytic mechanism.

  • 16.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Kinetic independence of the subunits of cytosolic glutathione transferase from the rat1985In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 231, no 2, p. 263-267Article in journal (Refereed)
    Abstract [en]

    The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.

  • 17.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism1988In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 250, no 3, p. 705-711Article in journal (Refereed)
    Abstract [en]

    Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.

  • 18. de Kloe, Gerdien E
    et al.
    Retra, Kim
    Geitmann, Matthis
    Källblad, Per
    Nahar, Tariq
    van Elk, René
    Smit, August B
    van Muijlwijk-Koezen, Jacqueline E
    Leurs, Rob
    Irth, Hubertus
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    de Esch, Iwan J P
    Surface Plasmon Resonance Biosensor Based Fragment Screening Using Acetylcholine Binding Protein Identifies Ligand Efficiency Hot Spots (LE Hot Spots) by Deconstruction of Nicotinic Acetylcholine Receptor α7 Ligands2010In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 53, no 19, p. 7192-7201Article in journal (Refereed)
    Abstract [en]

    The soluble acetylcholine binding protein (AChBP) is a homologue of the ligand-binding domain of the nicotinic acetylcholine receptors (nAChR). To guide future fragment-screening using surface plasmon resonance (SPR) biosensor technology as a label-free, direct binding, biophysical screening assay, a focused fragment library was generated based on deconstruction of a set of α7 nAChR selective quinuclidine containing ligands with nanomolar affinities. The interaction characteristics of the fragments and the parent compounds with AChBP were evaluated using an SPR biosensor assay. The data obtained from this direct binding assay correlated well with data from the reference radioligand displacement assay. Ligand efficiencies for different (structural) groups of fragments in the library were correlated to binding with distinct regions of the binding pocket, thereby identifying ligand efficiency hot spots (LE hot spots). These hot spots can be used to identity the most promising hit fragments in a large scale fragment library screen.

  • 19. Edalat, Maryam H
    et al.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Peptide phage display for probing GST-protein interactions.2005In: Methods Enzymol, ISSN 0076-6879, Vol. 401, p. 354-67Article in journal (Refereed)
    Abstract [en]

    Phage display is a powerful strategy for identifying protein-peptide interactions. Glutathione transferases (GSTs) play prominent roles in the cellular protection against oxidative stress by catalyzing detoxication reactions. In addition, GSTs seem to act in signaling pathways by means of interaction with other macromolecules such as protein kinases. This chapter describes how the technique of peptide phage display can be used to identify possible partners in GST-protein complexes.

  • 20.
    Edalat, Maryam
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Technology, Department of Materials Science. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Axelsson, Lars-Göran
    Selective expression of detoxifying glutathione transferases in mouse colon: effect of experimental colitis and the presence of bacteria.2004In: Histochem Cell Biol, ISSN 0948-6143, Vol. 122, no 2, p. 151-9Article in journal (Refereed)
  • 21.
    Eklund, Birgitta
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Elfarra, Adnan A.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Activation of anticancer prodrugs by human glutathione transferases2005In: FEBS Journal, Vol. 272, p. 546 Suppl-Article in journal (Refereed)
  • 22.
    Eklund, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Interpreting a Giant: Studies of Structure and Function of Tripeptidyl-peptidase II2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine peptidase that forms a gigantic homooligomeric complex, and is involved in the degradation of peptides in the cytosol. In addition, TPP II has been implicated in specific cellular processes, such as apoptosis control and adipogenesis, but if this is dependent on its endo- or exopeptidase activity remains to be determined. This work is devoted to the structure and function of TPP II, and to finding connections between the two.

    Evolutionarily conserved regions of TPP II have been identified, and sequence signatures have been constructed as an aid in identification of TPP II homologues. The conserved regions highlight amino acid residues of potential importance to structure, function or both. In addition, the first TPP II homologue in a prokaryote has been documented, which was likely the result of a horizontal gene transfer.

    Substrate binding for the exopeptidase activity of TPP II has been studied through mutagenesis of Glu-331, which revealed a molecular ruler mechanism that positions substrates for cleavage at the third peptide bond from the N-terminus. Thus, the well-known tripeptidyl-releasing property of TPP II could be explained. The exopeptidase activity was also probed by pH dependence studies, which revealed that a substrate with a smaller residue in the P1 position could bind non-productively to the active site. Furthermore, a difference in the pH dependence of KM between TPP II from Drosophila and homologues from mammals indicated a difference in the configuration of the binding pockets between these species.

    The endopeptidase activity of TPP II has also been investigated, and was found to differ from the exopeptidase activity. The endopeptidase activity appeared to be promiscuous and the preference for basic amino acid residues in the P1 position reported earlier could not be substantiated.

    In conclusion, many structural and mechanistic features have been observed in this work. This might be of value to future drug discovery efforts towards TPP II, and in elucidating the physiological role of this gigantic enzyme.

    List of papers
    1. Development, evaluation and application of tripeptidyl-peptidase II sequence signatures
    Open this publication in new window or tab >>Development, evaluation and application of tripeptidyl-peptidase II sequence signatures
    2009 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 484, no 1, p. 39-45Article in journal (Refereed) Published
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a cytosolic peptidase that has been implicated in fat formation and cancer, apparently independent of the enzymatic activity. In search for alternative functional regions, conserved motifs were identified and eleven signatures were constructed. Seven of the signatures covered previously investigated residues, whereas the functional importance of the other motifs is unknown. This provides directions for future investigations of alternative activities of TPP II. The obtained signatures provide an efficient bioinformatic tool for the identification of TPP II homologues. Hence, a TPP II sequence homologue from fission yeast, Schizosaccharomyces pombe, was identified and demonstrated to encode the TPP II-like protein previously reported as multicorn. Furthermore, an homologous protein was found in the prokaryote Blastopirellula marina, albeit the TPP II function was apparently not conserved. This gene is probably the result of a rare gene transfer from eukaryote to prokaryote.

    Keywords
    Serine protease, subtilase, sequence motif, cytosolic protein degradation, tripeptidyl-peptidase II
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-119529 (URN)10.1016/j.abb.2009.01.007 (DOI)000264927700006 ()19467630 (PubMedID)
    Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-12-12
    2. Investigation of a role for Glu-331 and Glu-305 in substrate binding of tripeptidyl-peptidase II
    Open this publication in new window or tab >>Investigation of a role for Glu-331 and Glu-305 in substrate binding of tripeptidyl-peptidase II
    2008 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1784, no 12, p. 1899-1907Article in journal (Refereed) Published
    Abstract [en]

    The aim of this study was to investigate the mechanism by which tripeptidyl-peptidase II (TPP II) can specifically release tripeptides from the free N-terminus of an oligopeptide. The subtilisin-like N-terminal part of TPP II was modelled using subtilisin as template. Two glutamate residues (Glu-305 and Glu-331) appeared to be positioned so as to interact with the positively charged N-terminus of the substrate. In order to test this potential interaction, both residues were replaced by glutamine and lysine. The catalytic efficiency was reduced 400-fold for the E331Q variant and 20000-fold for the E331K variant, compared with the wild-type (wt). A substantial part of this reduction was due to decreased substrate affinity, since the K(M) for both mutants was at least two orders of magnitude greater than for the wt. This decrease was linked specifically to interaction with the free N-terminal amino group, based on inhibition studies. Glu-305 appears to be essential for enzymatic activity, but the extremely low activity of the E305Q variant prevented an investigation of the involvement of Glu-305 in substrate binding. The present work is, to our knowledge, the first report to investigate a mechanism for a tripeptidyl-peptidase activity through site-directed mutagenesis.

    Keywords
    Tripeptidyl-peptidase II, Exopeptidase, Substrate specificity, Intracellular protein degradation, Oligomerization, Molecular ruler
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-100055 (URN)10.1016/j.bbapap.2008.08.017 (DOI)000261673300002 ()18822395 (PubMedID)
    Available from: 2009-03-24 Created: 2009-03-24 Last updated: 2017-12-13Bibliographically approved
    3. Inter-species variation in the pH dependence of tripeptidyl-peptidase II
    Open this publication in new window or tab >>Inter-species variation in the pH dependence of tripeptidyl-peptidase II
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a large enzyme complex (>4 MDa) participating in the general protein turn-over in the cell downstream of the proteasome. In addition, there have been reports of involvement of TPP II in different physiological situations. To facilitate further investigations of the physiological role of TPP II and its enzymatic properties, a characterization at protein level is necessary. Therefore, an expression system for murine TPP II using Escherichia coli has been developed. The pH-optimum for cleavage of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was investigated for mTPP II, and compared with human TPP II and TPP II from Drosophila melanogaster. It was shown that the mouse enzyme had similar pH dependence as the human enzyme, while dTPP II had a slightly lower optimum. Surprisingly, the investigation also demonstrated that TPP II from all sources showed a different pH-profile for hydrolysis of AAA-pNA compared to AAF-pNA. To investigate this observation further, steady-state kinetic parameters were determined at various pH. Since both the KM and Vmax are lower for cleavage of AAA-pNA, a potential explanation could be that the substrate AAA-pNA is non-productively bound to the active site of the enzyme.

    Keywords
    tripeptidyl-peptidase II, TPP II, AAF-pNA, AAA-pNA, steady-state kinetics, pH-dependence
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-134621 (URN)
    Available from: 2010-11-30 Created: 2010-11-30 Last updated: 2011-06-28
    4. Characterization of the endopeptidase activity of tripeptidyl-peptidase II
    Open this publication in new window or tab >>Characterization of the endopeptidase activity of tripeptidyl-peptidase II
    Show others...
    2012 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 424, no 3, p. 503-507Article in journal (Refereed) Published
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.

    Keywords
    TPP II, endopeptidase, alternate activity
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-134620 (URN)10.1016/j.bbrc.2012.06.144 (DOI)000307618800024 ()
    Available from: 2010-11-30 Created: 2010-11-30 Last updated: 2017-12-12Bibliographically approved
  • 23.
    Ekman, Pia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Eller, Marika
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Ragnarsson, Ulf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Two methods to avoid the effect of endogenous inhibitors during the assay of protein kinase C activity in tissue extracts.1992In: Preparative Biochemistry, ISSN 0032-7484, Vol. 22, no 2, p. 165-175Article in journal (Refereed)
    Abstract [en]

    Using H1 as substrate the protein kinase C activity of rat liver cell sap was increased about fourfold by treatment with DEAE-cellulose at pH 7.5 at an intermediate ionic strength due to removal of protein inhibitors. The activity of cell sap from rat spleen, brain or muscle was about doubled by the same treatment. In contrast, when a specific synthetic peptide substrate was used the corresponding increase of enzyme activity was not obtained when the inhibitors were removed. This shows that this type of substrates should be preferred for reliable assays of protein kinase C in crude extracts. The possible role of the protein inhibitors for the substrate specificity of protein kinase C is briefly discussed.

  • 24.
    Elfström, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Catalysis of potato epoxide hydrolase, StEH12005In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 390, p. 633-640Article in journal (Refereed)
    Abstract [en]

    The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 mmol·min-1·mg-1 with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower Km value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

  • 25.
    Elfström, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    The Saccharomyces cerevisiae ORF YNR064c protein has characteristics of an 'orphaned' epoxide hydrolase2005In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1748, p. 213-221Article in journal (Refereed)
    Abstract [en]

    The open reading frame YNR064c in Saccharomyces cerevisiae encodes a protein tentatively assigned as similar to a bacterialdehalogenase. In this study we conclude that the YNR064c protein displays characteristics of an epoxide hydrolase belonging to the a/hhydrolasefold family of enzymes. Endogenous expression of the protein in S. cerevisiae was confirmed and a His-tagged variant of theprotein was heterologously expressed in both Escherichia coli and Pichia pastoris for isolation and characterization. The YNR064c proteindisplayed low but reproducible epoxide hydrolase activity with racemic phenanthrene 9,10-oxide and trans- or cis-stilbene oxide.Phylogenetic analysis of related gene products found in various microorganisms suggested that the YNR064c protein is a member of a newsubclass of a/h-hydrolase fold enzymes.

  • 26.
    Elinder, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Geitmann, Matthis
    Gossas, Thomas
    Källblad, Per
    Winquist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Nordström, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Hämäläinen, Markkuu D.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology2011In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 16, no 1, p. 15-25Article in journal (Refereed)
    Abstract [en]

    A new fragment library for lead discovery has been designed and experimentally validated for use in surface plasmon resonance (SPR) biosensor-based screening. The 930 compounds in the library were selected from 4.6 million commercially available compounds using a series of physicochemical and medicinal chemistry filters. They were screened against 3 prototypical drug targets: HIV-1 protease, thrombin and carbonic anhydrase, and a nontarget: human serum albumin. compound solubility was not a problem under the conditions used for screening. The high sensitivity of the sensor surfaces allowed the detection of interactions for 35% to 97% of the fragments, depending on the target protein. None of the fragments was promiscuous (i.e., interacted with a stoichiometry ≥5:1 with all 4 proteins), and only 2 compounds dissociated slowly from all 4 proteins. The use of several targets proved valuable since several compounds would have been disqualified from the library on the grounds of promiscuity if fewer target proteins had been used. The experimental procedure allowed an efficient evaluation and exploration of the new fragment library and confirmed that the new library is suitable for SPR biosensor-based screening.

  • 27.
    Ellfolk, Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Isolation and properties of the CYP2D25 promoter: Transcriptional regulation by vitamin D3 metabolites2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 345, no 2, p. 568-572Article in journal (Refereed)
    Abstract [en]

    Previous studies have suggested that hepatic production of 25-hydroxyvitamin D3 may be suppressed by 1α,25-dihydroxyvitamin D3. However, the molecular details of these observations have not been clarified. In the current study, the 5´-flanking DNA sequence of CYP2D25, a porcine microsomal vitamin D 25-hydroxylase, was isolated and analyzed. The CYP2D25 promoter contains a putative vitamin D response element (VDRE). The promoter activity was markedly suppressed by 1α,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 in presence of vitamin D receptor (VDR). The data suggest that VDR-mediated inhibition of 25-hydroxylase(s) by vitamin D3 metabolites at the transcriptional level may play an important role in the regulation of 25-hydroxyvitamin D3 production in liver and other tissues.

  • 28.
    Fedulova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Experimental Conditions Affecting Functional Comparison of Highly Active Glutathione Transferases2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 413, no 1, p. 16-23Article in journal (Refereed)
    Abstract [en]

    Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. Combination of time-dependent and enzyme concentration-dependent loss of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurementsof GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.

  • 29.
    Fedulova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Raffalli-Mathieu, Francoise
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Characterization of porcine Alpha-class glutathione transferase A1-12011In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 507, no 2, p. 205-211Article in journal (Refereed)
    Abstract [en]

    An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissuesand animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence ofour clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to ourcloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1*. The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactionswas at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.

  • 30.
    Geitmann, Matthis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Dahl, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors2011In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 24, no 1, p. 60-70Article in journal (Refereed)
    Abstract [en]

    The mechanism and kinetics of the interactions between ligands and immobilized full-length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3-NS4A interaction consisted of a high-affinity (K(D) = 50 nM) and a low-affinity (K(D) = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism-based inhibitor VX 950 exhibited a time-dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN-191 showed no signs of time-dependent interactions, but ITMN-191 had the highest affinity of the tested compounds, with both the slowest dissociation (k(off)) and fastest association rate, closely followed by BILN 2061. The k(off) for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti-HCV lead discovery and optimization.

  • 31. Geitmann, Matthis
    et al.
    Elinder, Malin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Seeger, Christian
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Brandt, Peter
    de Esch, Iwan J P
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Identification of a Novel Scaffold for Allosteric Inhibition of Wild Type and Drug Resistant HIV-1 Reverse Transcriptase by Fragment Library Screening2011In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 3, p. 699-708Article in journal (Refereed)
    Abstract [en]

    A novel scaffold inhibiting wild type and drug resistant variants of human immunodeficiency virus type 1 reverse transcriptase (HIV-1RT) has been identified in a library consisting of 1040 fragments. The fragments were significantly different from already known non-nucleoside reverse transcriptase inhibitors (NNRTIs), as indicated by a Tversky similarity analysis. A screening strategy involving SPR biosensor-based interaction analysis and enzyme inhibition was used. Primary biosensor-based screening, using short concentration series, was followed by analysis of nevirapine competition and enzyme inhibition, thus identifying inhibitory fragments binding to the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Ten hits were discovered, and their affinities and resistance profiles were evaluated with wild type and three drug resistant enzyme variants (K103N, Y181C, and L100I). One fragment exhibited submillimolar K(D) and IC(50) values against all four tested enzyme variants. A substructure comparison between the fragment and 826 structurally diverse published NNRTIs confirmed that the scaffold was novel. The fragment is a bromoindanone with a ligand efficiency of 0.42 kcal/mol(-1).

  • 32. Geitmann, Matthis
    et al.
    Retra, Kim
    de Kloe, Gerdien E
    Homan, Evert
    Smit, August B
    de Esch, Iwan J P
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Interaction kinetic and structural dynamic analysis of ligand binding to acetylcholine-binding protein2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 37, p. 8143-8154Article in journal (Refereed)
    Abstract [en]

    The mechanism of agonist interactions with Cys-loop ligand-gated ion channels has been studied using the acetylcholine-binding protein (AChBP) from Lymnaea stagnalis as a model protein, and acetylcholine, nicotine, epibatidine and a series of substituted quinuclidines as ligands. A biosensor-based assay for direct interaction studies of immobilized AChBP and small molecule ligands was developed. It allowed the characterization of the interaction kinetics of the ligands and the structural dynamics of the protein. The interactions with AChBP were very sensitive to variations in the experimental conditions and showed several types of complexities. These could be resolved into two types of ligand-induced secondary effects with different kinetics, representing fast and slow conformational changes. The data could be rationalized in a mechanistic model and a structural interpretation of the interaction was obtained by molecular modelling involving induced-fit and loop flexibility simulations. The data suggests that AChBP exhibits ligand-induced structural dynamics, as expected for the ligand gating mechanism of Cys-loop receptors. It shows that the formation of the initial encounter complex between AChBP and ligands is very rapid, in accordance with the functional characteristics required of neurotransmission. These developed procedures will enable further exploration of the mechanism of Cys-loop receptor function and the identification of specific ligands suitable for pharmacological use.

  • 33.
    Ghasemzadeh, Nasim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tore W. Wilhelmsen, Tore W.
    Norwegian Medicines Agency.
    Nyberg, Fred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hjertén, Stellan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics2010In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 31, no 16, p. 2722-2729Article in journal (Refereed)
    Abstract [en]

    Although protein biomarkers have a great potential as biomarkers for diagnosis of diseases, they are seldom used in hospitals. There are many reasons for this, for instance, the difficulties to (i) find a biomarker for which the concentration in body fluids clearly differs between patients and healthy subjects, (ii) attain purification of the biomarker close to 100%, which is required for production of conventional protein antibodies as well as artificial gel antibodies for selective capture of a biomarker, (iii) design a standard curve for rapid and accurate determination of the concentration of the biomarker in the body fluid because of adsorption of the biomarker onto vials, pipettes, etc., (iv) determine accurately the sample volume delivered by a pipette, (v) avoid polymerization of the biomarker upon storage and to decide whether it is in the form not only of monomers, but also of dimers, trimers, etc., in the native state, (vi) determine the degree of possible glycosylation and amidation of the biomarker and (vii) decide whether glycosylation and amidation positively or negatively affects the possibility to use the protein as a biomarker. In this article, we discuss in quantitative terms the difficulties (iii-vii) and how to overcome them, which also may help to overcome the difficulty (ti), which in turn minimizes difficulty (i).

  • 34.
    Gogoll, Adolf
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Oscarsson, Sven
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Reaction of pyridine derivatives with butyl glycidyl ether as a model system for glycidyl ether modified agarose: structural assignment by selective inept spectra1990In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 46, no 7, p. 2539-2548Article in journal (Refereed)
    Abstract [en]

    The reaction of 2-thio-pyridine N-oxide, 2-amino-, 2-hydroxy, 2-thio-, and 4-thiopyridine with butyl glycidyl ether was investigated as a model system for the functionalization of 2,3-epoxypropyl activated agarose. Unambiguous structural assignment of the products was provided by selective INEPT and nuclear Overhauser difference spectra. All reactions were shown to give only one of the possible regioisomers. Further conclusions regarding the structure of the agarose derivatives were drawn from IR spectra.

  • 35.
    Gustafsson, Sofia S.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Vrang, Lotta
    Terelius, Ylva
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Quantification of interactions between drug leads and serum proteins by use of "binding efficiency"2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 409, no 2, p. 163-175Article in journal (Refereed)
    Abstract [en]

    To develop efficient and reliable methods for prediction of serum protein binding of drug leads, the kinetic characteristics for the interactions between selected compounds and human serum albumin and α(1)-acid glycoprotein have been explored using a surface plasmon resonance biosensor. Conventional methods for quantification of interactions (i.e., using rate constants or affinities determined on the basis of a reasonable mechanistic model) were applicable for only a few of the compounds. The affinity of a primary interaction and the contribution of lower affinity secondary interactions could be estimated for some compounds, but the affinity of many compounds could not be quantified by either of these methods. To have a quantification method that could be used for all compounds, independent of affinity and complexity of interaction mechanisms, the concept of "binding efficiency," analogous to "catalytic efficiency" used for enzymes, was developed. It allowed the quantification of the binding of compounds interacting with weak affinity and for which saturation is not reached within a concentration range where the compound is soluble or when the influence of interactions with secondary sites makes interpretations difficult. In addition, compounds with large fractional binding can be identified by this strategy and simply quantified relative to reference compounds. This approach will enable ranking and identification of structure-activity relationships of compounds with respect to their serum protein binding profile.

  • 36.
    Gutierrez Arenas, Omar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Sensitivity analysis and error structure of progress curves2006In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 358, no 1, p. 1-10Article in journal (Refereed)
    Abstract [en]

    Both the sensitivity of the monitored signal in progress curves to variations in enzyme concentration and the standard deviation of this signal were analyzed as a function of the proportion of transformed substrate. Three enzymes catalyzing essentially irreversible reactions were used as model systems: HIV-1 protease, glutathione reductase, and glutathione transferase. For all enzymes analyzed, the sensitivity was maximal when 60-80% of the substrate had been transformed. The standard deviation of reaction progress curve data replicates was also maximal at these substrate conversion levels, a result that was attributed to the influence of the sensitivity to random dispersion of the enzyme concentration. On this basis, we developed a model for the standard deviation of reaction progress curves that gave a good description of the experimental data and efficiently reduced the heteroscedasticity of residuals in a weighted fit of progress curves. This standard deviation model can be used for obtaining more efficient parameter estimates, to simulate noise in Monte Carlo procedures, and to delineate detection limits of enzyme inhibition. The transient increases in the sensitivity and in the standard deviation in progress curves are proposed to be features common to most enzymatic assays.

  • 37. Hederos, Sofia
    et al.
    Broo, Kerstin
    Jakobsson, Emma
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Kleywegt, Gerard J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mannervik, Bengt
    Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Baltzer, Lars
    Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    A new enzyme by rational design - the incorporation of a single His residue enables efficient thioester hydrolysis by human glutathione transferase A1-12004In: Proc. Nat. Acad. Sci., Vol. 101, p. 13163-13167Article in journal (Refereed)
    Abstract [en]

    A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.

  • 38. Hederos, Sofia
    et al.
    Broo, Kerstin S
    Jakobsson, Emma
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. ICM.
    Kleywegt, Gerard J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. ICM.
    Mannervik, Bengt
    Department of Biochemistry. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Baltzer, Lars
    Department of Chemistry. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Incorporation of a single His residue by rational design enables thiol-ester hydrolysis by human glutathione transferase A1-1.2004In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 101, no 36, p. 13163-7Article in journal (Refereed)
    Abstract [en]

    A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.

  • 39.
    Hegazy, Usama
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Site-directed substitution of the key residue at the subunit interface of GST P1-1 by S-alkylcysteine residues sustains glutathione binding and catalytic activity of the enzyme2005In: FEBS Journal, Vol. 272, p. 87 Suppl-Article in journal (Refereed)
  • 40.
    Ivarsson, Ylva
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Exploring the importance of an active site residue on the stereo and regio selectivity of Mu class glutathione transferases2005In: FEBS Journal, Vol. 272, p. 88 Suppl-Article in journal (Refereed)
  • 41.
    Ivarsson, Ylva
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Regio- and enantioselectivities in epoxide conjugations are modulated by residue 210 in Mu class glutathione transferases.2005In: Protein Eng Des Sel, ISSN 1741-0126, Vol. 18, no 12, p. 607-16Article in journal (Refereed)
    Abstract [en]

    The homologous human glutathione transferases (GSTs) M1-1 and M2-2 have similar catalytic activities with many electrophilic substrates, but differ strikingly in their conjugation of epoxides with glutathione. Residue 210, Thr in GST M2-2 and Ser in GST M1-1, is a key active-site component in determining the activity profile with epoxide substrates. This residue is hypervariable in Mu class GSTs, suggesting that it has special significance in the evolution of new functions. The present study shows that minor modifications of this residue can have major consequences for the enzyme-catalyzed epoxide conjugations. In general, a Ser at position 210 gives the highest catalytic efficiency, but the relatively high activity with an Ala placed on this position demonstrates that a hydroxyl group is not required. In contrast, a Thr residue suppresses the activity with epoxides by several orders of magnitude without major effects on the activity with alternative GST substrates. Residue 210 influences both the regio- and enantioselectivity with chiral and prochiral epoxides of stilbene and styrene and influences the distribution of isomeric glutathione conjugates. Thus, residue 210 contributes to both stereoselective recognition of the substrates and to partitioning of the isomeric reactants to the alternative transition states leading to separate chiral products.

  • 42.
    Ivarsson, Ylva
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Norrgård, Malena A
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Tars, Kaspar
    Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    A positively selected residue influences enzyme functionalities2006In: The FASEB Journal, Vol. 20, p. A474-Article, review/survey (Other (popular scientific, debate etc.))
  • 43.
    Johansson, Emma
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Engvall, Caroline
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Arfvidsson, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Lundahl, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Development and initial evaluation of PEG-stabilized disks as novel model membranes2005In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 113, no 2, p. 183-192Article in journal (Refereed)
    Abstract [en]

    We show in this study that stable dispersions dominated by flat bilayer disks may be prepared from a carefully optimized mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] [PEG-DSPE(5000)]. By varying the content of the latter component, the average diameter of the disks can be changed in the interval from about 15 to 60 nm. The disks show excellent long-term stability, and their size and structure remain unaltered in the temperature range between 25 and 37 degrees C. The utility of the disks as artificial model membranes was confirmed and compared to uni- and multilamellar liposomes in a series of drug partition studies. Data obtained by isothermal titration calorimetry and drug partition chromatography (also referred to as immobilized liposome chromatography) indicate that the bilayer disks may serve as an attractive and sometimes superior alternative to liposomes in studies aiming at the investigation of drug-membrane interactions. The disks may, in addition, hold great potential for structure/function studies of membrane-bound proteins. Furthermore, we suggest that the sterically stabilized bilayer disks may prove interesting as carriers for in vivo delivery of protein/peptide, as well as conventional amphiphilic and/or hydrophobic, drugs.

  • 44.
    Jonas, Kristina
    et al.