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Mannervik, Bengt
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Publications (10 of 63) Show all publications
Zhang, W., Dourado, D. F. A. & Mannervik, B. (2015). Evolution of the active site of human glutathione transferase A2-2 for enhanced activity with dietary isothiocyanates. Biochimica et Biophysica Acta - General Subjects, 1850(4), 742-749
Open this publication in new window or tab >>Evolution of the active site of human glutathione transferase A2-2 for enhanced activity with dietary isothiocyanates
2015 (English)In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1850, no 4, p. 742-749Article in journal (Refereed) Published
Abstract [en]

Background: Organic isothiocyanates (ITCs) are produced by plants, in which they are released from glucosinolates by myrosinase. ITCs are generally toxic and serve as a chemical defense against herbivorous insects and against infections by microorganisms. In mammalian tissues subtoxic concentrations of ITCs can provide protective effects against cancer and other diseases partially by induction of glutathione transferases (GSTs) and other detoxication enzymes. Thus, human consumption of edible plants rich in ITCs is presumed to provide health benefits. ITCs react with intracellular glutathione to form dithiocarbamates, catalyzed by GSTs. Formation of glutathione conjugates is central to the biotransformation of ITCs and leads to a route for their excretion. Clearly, the emergence of ITC conjugating activity in GSTs is essential from the biological and evolutionary perspective. Methods: In the present investigation an active-site-focused mutant library of GST A2-2 has been screened for enzyme variants with enhanced ITC activity. Results: Significantly superior activities were found in 34 of the approximately 2000 mutants analyzed, and the majority of the superior GSTs featured His and Gly residues in one of the three active-site positions subjected to mutagenesis. Conclusions: We explored the propensity of GSTs to obtain altered substrate selectivity and moreover, identified a specific pattern of mutagenesis in GST for enhanced PEITC detoxification, which may play an important role in the evolution of adaptive responses in organisms subjected to ITCs. General significance: The facile acquisition of enhanced ITC activity demonstrates that this important detoxication function can be promoted by numerous evolutionary trajectories in sequence space. (C) 2014 Elsevier B.V. All rights reserved.

Keywords
Glutathione transferase, Isothiocyanate, Directed evolution, Enzyme engineering, Detoxication
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-248424 (URN)10.1016/j.bbagen.2014.12.021 (DOI)000350191700018 ()25542299 (PubMedID)
Available from: 2015-04-10 Created: 2015-03-30 Last updated: 2017-12-04Bibliographically approved
Kjellander, M., Mazari, A. M. .., Boman, M., Mannervik, B. & Johansson, G. (2014). Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening and stability. Analytical Biochemistry, 446, 59-63
Open this publication in new window or tab >>Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening and stability
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2014 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 446, p. 59-63Article in journal (Refereed) Published
Abstract [en]

The previously uncharacterized Drosophila melanogaster Epsilon class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via ∊-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that for enzymes in solution and no leakage of enzyme from the matrix could be observed.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-210099 (URN)10.1016/j.ab.2013.10.004 (DOI)000329949500010 ()
Available from: 2013-10-31 Created: 2013-10-31 Last updated: 2017-12-06Bibliographically approved
Dourado, D. F. A., Fernandes, P. A., Mannervik, B. & Ramos, M. J. (2014). Isomerization of Delta(5)-Androstene-3,17-dione into Delta(4)-Androstene-3, 17-dione Catalyzed by Human Glutathione Transferase A3-3: A Computational Study Identifies a Dual Role for Glutathione. Journal of Physical Chemistry A, 118(31), 5790-5800
Open this publication in new window or tab >>Isomerization of Delta(5)-Androstene-3,17-dione into Delta(4)-Androstene-3, 17-dione Catalyzed by Human Glutathione Transferase A3-3: A Computational Study Identifies a Dual Role for Glutathione
2014 (English)In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 118, no 31, p. 5790-5800Article in journal (Refereed) Published
Abstract [en]

Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond isomerization of Delta 5-androstene-3,17-dione (Delta(5)-AD) and Delta(5)-pregnene-3,20-dione (Delta(5)-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Delta(5)-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Delta(5)-AD C4 atom to the Delta(5)-AD C6 atom, with a rate limiting activation energy of 15.8 kcal.mol(-1). GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the 03 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leul11, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Delta(4)-AD crystal structure and is consistent with all available experimental data.

National Category
Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-230947 (URN)10.1021/jp410810q (DOI)000340222500008 ()
Available from: 2014-09-03 Created: 2014-09-01 Last updated: 2017-12-05Bibliographically approved
Zhang, W. & Mannervik, B. (2013). An improved dual-tube megaprimer approach for multi-site saturation mutagenesis. World Journal of Microbiology & Biotechnology, 29(4), 667-672
Open this publication in new window or tab >>An improved dual-tube megaprimer approach for multi-site saturation mutagenesis
2013 (English)In: World Journal of Microbiology & Biotechnology, ISSN 0959-3993, E-ISSN 1573-0972, Vol. 29, no 4, p. 667-672Article in journal (Refereed) Published
Abstract [en]

Saturation mutagenesis is a powerful tool in protein engineering. Even though QuikChange site-directed mutagenesis method is dominantly used in laboratories, it could not be successfully applied to the generation of a focused mutant library of human glutathione transferase A2-2. In the present study, we further developed an improved versatile dual-tube approach of randomizing difficult-to-amplify targets, exhibiting significant improvement towards equal distribution of nucleotides at randomized sites compared to other published methods.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-158396 (URN)10.1007/s11274-012-1222-z (DOI)000316291000010 ()
Available from: 2011-09-06 Created: 2011-09-06 Last updated: 2017-12-08Bibliographically approved
Sun, S., Zhang, W., Mannervik, B. & Andersson, D. I. (2013). Evolution of Broad Spectrum beta-Lactam Resistance in an Engineered Metallo-beta-lactamase. Journal of Biological Chemistry, 288(4), 2314-2324
Open this publication in new window or tab >>Evolution of Broad Spectrum beta-Lactam Resistance in an Engineered Metallo-beta-lactamase
2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 4, p. 2314-2324Article in journal (Refereed) Published
Abstract [en]

The extensive use and misuse of antibiotics during the last seven decades has led to the evolution and global spread of a variety of resistance mechanisms in bacteria. Of high medical importance are beta-lactamases, a group of enzymes inactivating beta-lactam antibiotics. Metallo-beta-lactamases (MBLs) are particularly problematic because of their ability to act on virtually all classes of beta-lactam antibiotics. An engineered MBL (evMBL9) characterized by low level activity with several beta-lactam antibiotics was constructed and employed as a parental MBL in an experiment to examine how an enzyme can evolve toward increased activity with a variety of beta-lactam antibiotics. We designed and synthesized a mutant library in which the substrate activity profile was varied by randomizing six active site amino acid residues. The library was expressed in Salmonella typhimurium, clones with increased resistance against seven different beta-lactam antibiotics (penicillin G, ampicillin, cephalothin, cefaclor, cefuroxime, cefoperazone, and cefotaxime) were isolated, and the MBL variants were characterized. For the majority of the mutants, bacterial resistance was significantly increased despite marked reductions in both mRNA and protein levels relative to those of parental evMBL9, indicating that the catalytic activities of these mutant MBLs were highly increased. Multivariate analysis showed that the majority of the mutant enzymes were generalists, conferring increased resistance against most of the examined beta-lactams.

National Category
Medical and Health Sciences Natural Sciences
Identifiers
urn:nbn:se:uu:diva-196042 (URN)10.1074/jbc.M112.430199 (DOI)000314211500021 ()
Available from: 2013-03-04 Created: 2013-03-04 Last updated: 2017-12-06Bibliographically approved
Musdal, Y., Hegazy, U. M., Aksoy, Y. & Mannervik, B. (2013). FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1. Chemico-Biological Interactions, 205(1), 53-62
Open this publication in new window or tab >>FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1
2013 (English)In: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 205, no 1, p. 53-62Article in journal (Refereed) Published
Abstract [en]

Objective: Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. Methods: We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. Results: We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. Conclusion and practical implications: In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords
Glutathione transferase P1-1, FDA-approved drugs, Enzyme inhibition, Adjuvant chemotherapeutics, Ethacrynic acid
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-220559 (URN)10.1016/j.cbi.2013.06.003 (DOI)000331169900009 ()
Available from: 2014-03-20 Created: 2014-03-17 Last updated: 2017-12-05Bibliographically approved
Shibata, A., Nakano, Y., Ito, M., Araki, M., Zhang, J., Yoshida, Y., . . . Abe, H. (2013). Fluorogenic probes using 4-substituted-2-nitrobenzenesulfonyl derivatives as caging groups for the analysis of human glutathione transferase catalyzed reactions. The Analyst, 138(24), 7326-7330
Open this publication in new window or tab >>Fluorogenic probes using 4-substituted-2-nitrobenzenesulfonyl derivatives as caging groups for the analysis of human glutathione transferase catalyzed reactions
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2013 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 138, no 24, p. 7326-7330Article in journal (Refereed) Published
Abstract [en]

We have synthesized a series of 4-substituted-2-nitrobenzene-sulfonyl compounds for caged fluorogenic probes and conducted a Hammett plot analysis using the steady-state kinetic parameters. The results revealed that the glutathione transferase (GST) alpha catalyzed reaction was dependent on the sigma value in the same way as the non-enzymatic reaction, whereas the dependence of the sigma value of the GST mu and pi was not as pronounced as that of GST alpha.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-213096 (URN)10.1039/c3an01339a (DOI)000326988500009 ()
Available from: 2013-12-19 Created: 2013-12-18 Last updated: 2017-12-06Bibliographically approved
Dourado, D. F. A., Fernandes, P. A., Ramos, M. J. & Mannervik, B. (2013). Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA). Biochemistry, 52(45), 8069-8078
Open this publication in new window or tab >>Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA)
2013 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 45, p. 8069-8078Article in journal (Refereed) Published
Abstract [en]

Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO- groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-219167 (URN)10.1021/bi4005705 (DOI)000330017700019 ()
Available from: 2014-02-26 Created: 2014-02-24 Last updated: 2017-12-05Bibliographically approved
Mannervik, B. (2012). Five Decades with Glutathione and the GSTome. Journal of Biological Chemistry, 287(9), 6072-6083
Open this publication in new window or tab >>Five Decades with Glutathione and the GSTome
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 9, p. 6072-6083Article in journal (Other academic) Published
Abstract [en]

Uncle Folke inspired me to become a biochemist by demonstrating electrophoresis experiments on butterfly hemolymph in his kitchen. Glutathione became the subject for my undergraduate project in 1964 and has remained a focal point in my research owing to its multifarious roles in the cell. Since the 1960s, the multiple forms of glutathione transferase (GST), the GSTome, were isolated and characterized, some of which were discovered in our laboratory. Products of oxidative processes were found to be natural GST substrates. Examples of toxic compounds against which particular GSTs provide protection include 4-hydroxynonenal and ortho-quinones, with possible links to the etiology of Alzheimer and Parkinson diseases and other degenerative conditions. The role of thioltransferase and glutathione reductase in the cellular reduction of disulfides and other oxidized forms of thiols was clarified. Glyoxalase I catalyzes still another glutathione-dependent detoxication reaction. The unusual steady-state kinetics of this zinc-containing enzyme initiated model discrimination by regression analysis. Functional properties of the enzymes have been altered by stochastic mutations based on DNA shuffling and rationally tailored by structure-based redesign. We found it useful to represent promiscuous enzymes by vectors or points in multidimensional substrate-activity space and visualize them by multivariate analysis. Adopting the concept "molecular quasi-species," we describe clusters of functionally related enzyme variants that may emerge in natural as well as directed evolution.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-171671 (URN)10.1074/jbc.X112.342675 (DOI)000300791800001 ()
Available from: 2012-03-27 Created: 2012-03-25 Last updated: 2017-12-07Bibliographically approved
Zhang, W., Dourado, D. F. A., Fernandes, P., Ramos, M. & Mannervik, B. (2012). Multidimensional epistasis and fitness landscapes in enzyme evolution. Biochemical Journal, 445, 39-46
Open this publication in new window or tab >>Multidimensional epistasis and fitness landscapes in enzyme evolution
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2012 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 445, p. 39-46Article in journal (Refereed) Published
Abstract [en]

The conventional analysis of enzyme evolution is to regard one single salient feature as a measure of fitness, expressed in a milieu exposing the possible selective advantage at a given time and location. Given that a single protein may serve more than one function, fitness should be assessed in several dimensions. In the present study we have explored individual mutational steps leading to a triple-point-mutated human GST (glutathione transferase) A2-2 displaying enhanced activity with azathioprine. A total of eight alternative substrates were used to monitor the diverse evolutionary trajectories. The epistatic effects of the imitations on catalytic activity were variable in sign and magnitude and depended on the substrate used, showing that epistasis is a multidimensional quality. Evidently, the multidimensional fitness landscape can lead to alternative trajectories resulting in enzymes optimized for features other than the selectable markers relevant at the origin of the evolutionary process. In this manner the evolutionary response is robust and can adapt to changing environmental conditions.

Keywords
epistasis, evolutionary trajectories, fitness landscape, multivariate data analysis, protein evolution, substrate selectivity
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-158397 (URN)10.1042/BJ20120136 (DOI)000306874300004 ()
Note

Original manuscript title: "Epistasis is a multidimensional property in the evolutionary trajectories of glutathione transferase in alternative-substrate-activity space"

Available from: 2011-09-06 Created: 2011-09-06 Last updated: 2017-12-08Bibliographically approved
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