Profiles

Bengt Mannervik

Bengt Mannervik

Professor

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Arbetar vid Institutionen för neurokemi
Telefon 08-16 18 82
E-post bengt.mannervik@neurochem.su.se
Besöksadress Svante Arrheniusväg 16 B
Rum C450
Postadress Institutionen för neurokemi 106 91 Stockholm

Om mig

 

Professor of Biochemistry

Adjunct Professor in Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA

 

RESEARCH PROJECTS

Enzymes: from the test tube into the cell     

(Supported by the Swedish Research Council)

Enzymology is a cornerstone in the molecular life sciences. Rates and specificities of chemical reactions in cells as well as cellular signaling are governed by enzymes. Besides providing fundamental understanding of the dynamics of life processes, enzymology has essential applications in drug discovery and biotechnology. In addition, it is becoming increasingly evident that enzymes are integral components of more wide-ranging molecular interactions such that the study of enzyme functions has to be extended into their cellular context. Based on the cutting-edge advances in molecular life sciences, we will move enzyme research from the test tube into the cell. Genes encoding enzymes in different variants will be chemically synthesized, purified enzyme proteins will be produced and inserted directly into cells, or expressed intracellularly from transfected DNA. The projected research encompasses studies at different levels of complexity:

  • In vitro evolution and characterization of novel enzyme functions
  • Incorporation of enzymes into cells in culture
  • Modifying the enzyme composition of multicellular organisms

The first level encompasses directed evolution of enzymes for activity against targeted substrates such as environmental contaminants. Included are also studies of the development of drugs that attenuate hormone production.

The second level involves the effects of introducing enzymes into cells studied by both proteomics and transcriptomics. In particular, the modulation of cellular signaling via steroid hormones is addressed.

The third level comprises studies of transgenic fruit flies as well as plants expressing enzymes that confer designated properties to the organism. Acquisition of resistance to natural and synthetic toxic compounds and development of plants for bioremediation are important applications in medicine and biotechnology

Molecular toolbox of therapeutic enzymes for treatment of childhood cancers

(Supported by the Swedish Childhood Cancer Foundation)

We are designing combinations of alternative drug-activating enzymes linked to different tumor-targeting antibodies, which could be expected to give synergy and enhanced therapeutic efficacy in the treatment of leukemia and other malignancies. Human glutathione transferase (GST) enzymes are engineered for increased catalytic activity with a prodrug with the aim of delivering the enzyme selectively to a tumor. Antibodies fused with the GST enzymes will bind to epitopes of the targeted cancer cells, where the accompanying enzyme will exert its drug-activating function. For proof-of-concept our prototype for recombinant GST-antibody proteins is based on high affinity for the epitope CD19, which is overexpressed in malignant tumors of the B-cell lineage, including acute lymphoblastic leukemia (ALL). Our GST-antibody fusion proteins are obtained by chemical synthesis of DNA encoding the desired construct followed by heterologous protein expression and purification. Antibodies by themselves have tumor-killing properties, and the simultaneous prodrug activation by the bound GST is expected to offer synergy and increased tumor cell death. Our novel approach of GST-mediated toxicity directed to CD19-expressing cancer cells synergistically combines the effects of conventional chemotherapy with those of immunotherapy, which will reduce the likelihood of relapse of disease as well as counteract secondary malignancies due to off-target effects. The approach is generic and could be implemented with alternative binding proteins and GSTs with different functionalities, thereby establishing a molecular toolbox for alternative treatments.

 

Biosynthesis of dendrogenin A, a potent tumor-suppressing cholesterol metabolite

(Supported by the Swedish Cancer Society)

Dendrogenin A (DDA), which serves as a suppressor of oncogenesis and induces re-differentiation of cancer cells in mammalian tissues, is a product of the enzymatic conjugation of 5,6α-epoxy-cholesterol with histamine. The nature of the enzyme is so far unknown, but unpublished data suggest that a glutathione transferase (GST) is the biocatalyst. Based on our GST expertise and our collection of human and other mammalian GST enzymes (Mannervik, 2012) we will investigate the nature of the biocatalyst(s) accountable for DDA formation.

  • A convenient novel method to assay enzymatic DDA formation will be developed.
  • The human genome encodes 17 soluble GSTs and the GST enzyme(s) catalyzing DDA formation will be identified.
  • The catalytic mechanism will be determined and the possible interference with DDA synthesis by conventional GST activities elucidated.
  • The possible effect of tamoxifen and other drugs on DDA formation will be studied.
  • Approaches to optimize DDA formation in vivo will be pursued.
  • A procedure for enzymatic largescale synthesis of DDA will be developed.

Originally obtained by organic synthesis, dendrogenin A (DDA) is the first “steroidal alkaloid” to be identified in human tissues. DDA is generated from the stereoselective enzymatic conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine (HA). By contrast, the stereoisomeric 5,6β-EC is not conjugated.

Glutathione transferases provide resistance to chemical stress in rice (Oryza sativa)

(Collaboration with National Research Center, Cairo, Egypt)

The overall purpose of the project is to develop the biochemical and biotechnical tools to improve the resistance of cultivated rice to environmental challenges and to herbicides. In this work cutting-edge protein engineering will be established in the Egyptian institution that subsequently can be applied to various areas of biotechnology. From the Swedish side the project presents an opportunity to engage in an application of enzymology to solve societal problems arising in the wake of climate change.

The basis for the present investigation is a family of glutathione transferase (GST) enzymes, in which certain members serve to defend the organism against toxic insults. We propose to identify key protective GST enzymes in rice and improve their useful properties by directed evolution. The final outcome of the work is foreseen to be a basis for breeding or creating improved strains of rice, which can overcome the emerging problems of decreased production and lead to improved yields in Egypt and other countries producing this crop.

 

Publikationer

I urval från Stockholms universitets publikationsdatabas
  • 2017. Kyriakos Tzafestas (et al.). New Phytologist 214 (1), 294-303

    The explosive 2,4,6-trinitrotoluene (TNT) is a significant, global environmental pollutant that is both toxic and recalcitrant to degradation. Given the sheer scale and inaccessible nature of contaminated areas, phytoremediation may be a viable clean-up approach. Here, we have characterized a Drosophila melanogaster glutathione transferase (DmGSTE6) which has activity towards TNT. Recombinantly expressed, purified DmGSTE6 produces predominantly 2-glutathionyl-4, 6-dinitrotoluene, and has a 2.5-fold higher Maximal Velocity (Vmax), and five-fold lower Michaelis Constant (Km) than previously characterized TNT-active Arabidopsis thaliana (Arabidopsis) GSTs. Expression of DmGSTE6 in Arabidopsis conferred enhanced resistance to TNT, and increased the ability to remove TNT from contaminated soil relative to wild-type plants. Arabidopsis lines overexpressing TNT-active GSTs AtGST-U24 and AtGST-U25 were compromised in biomass production when grown in the absence of TNT. This yield drag was not observed in the DmGSTE6-expressing Arabidopsis lines. We hypothesize that increased levels of endogenous TNT-active GSTs catalyse excessive glutathionylation of endogenous substrates, depleting glutathione pools, an activity that DmGST may lack. In conclusion, DmGSTE6 has activity towards TNT, producing a compound with potential for further biodegradation. Selecting or manipulating plants to confer DmGSTE6-like activity could contribute towards development of phytoremediation strategies to clean up TNT from polluted military sites.

  • 2016. Bengt Mannervik, Birgitta Sjödin. Pharmaceutical Bioprocessing 4 (2), 25-30

    6-Chloro- and 6-bromopurines can cross the blood-brain barrier and in situ give rise to substrates of multidrug resistance-associated proteins (MRPs). The electrophilic purines form glutathione conjugates in reactions catalyzed by intracellular glutathione transferases (GSTs), and the conjugates are subsequently exported from the cells by ATP-dependent membrane transporters. In rodent model systems it has been demonstrated that suitably radiolabeled 6-halogenopurines by this scheme are pro-probes useful in monitoring the functionality of MRPs in intact brains using positron emission tomography. Prior to applications in human subjects it is imperative to establish the purine pro-probes as effective substrates for human GSTs occurring in brain and other tissues. We have developed a spectrophotometric assay for the glutathione conjugation and determined specific activities with a range of human GSTs as well as some rat GSTs for comparison. The ubiquitous GST P1-1 showed the highest activities with the 6-halogenopurines, which bodes well for the application of pro-probes for human investigations.

  • 2017. Yaman Musdal, Sridhar Govindarajan, Bengt Mannervik. Protein Engineering Design & Selection 30 (8), 543-549

    Exploring the vicinity around a locus of a protein in sequence space may identify homologs with enhanced properties, which could become valuable in biotechnical and other applications. A rational approach to this pursuit is the use of 'infologs', i.e. synthetic sequences with specific substitutions capturing maximal sequence information derived from the evolutionary history of the protein family. Ninety-five such infolog genes of poplar glutathione transferase were synthesized and expressed in Escherichia coli, and the catalytic activities of the proteins determined with alternative substrates. Sequence-activity relationships derived from the infologs were used to design a second set of 47 infologs in which 90% of the members exceeded wild-type properties. Two mutants, C2 (V55I/E95D/D108E/A160V) and G5 (F13L/C70A/G122E), were further functionally characterized. The activities of the infologs with the alternative substrates 1-chloro-2,4-dinitrobenzene and phenethyl isothiocyanate, subject to different chemistries, were positively correlated, indicating that the examined mutations were affecting the overall catalytic competence without major shift in substrate discrimination. By contrast, the enhanced protein expressivity observed in many of the mutants were not similarly correlated with the activities. In conclusion, small libraries of well-defined infologs can be used to systematically explore sequence space to optimize proteins in multidimensional functional space.

  • 2015. Sridhar Govindarajan (et al.). ACS Synthetic Biology 4 (3), 221-227

    We have used design of experiments (DOE) and systematic variance to efficiently explore glutathione transferase substrate specificities caused by amino acid substitutions. Amino acid substitutions selected using phylogenetic analysis were synthetically combined using a DOE design to create an information-rich set of gene variants, termed infologs. We used machine learning to identify and quantify protein sequence-function relationships against 14 different substrates The resulting models were quantitative and predictive, serving as a guide for engineering of glutathione transferase activity toward a diverse set of herbicides Predictive quantitative models like those presented here have broad applicability for bioengineering.

  • 2012. Bengt Mannervik. Journal of Biological Chemistry 287 (9), 6072-6083

    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.

  • 2012. Wei Zhang (et al.). Biochemical Journal 445, 39-46

    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.

Visa alla publikationer av Bengt Mannervik vid Stockholms universitet

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Senast uppdaterad: 17 november 2017

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