| 1. |
- Musdal, Yaman, et al.
(författare)
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Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants
- 2017
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 30:8, s. 543-549
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Kurtovic, Sanela, et al.
(författare)
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Structural determinants of glutathione transferases with azathioprine activity identified by DNA shuffling of alpha class members
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 375:5, s. 1365-1379
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Tidskriftsartikel (refereegranskat)abstract
- A library of alpha class glutathione transferases (GSTs), composed of chimeric enzymes derived from human (A1-1, A2-2 and A3-3), bovine (A1-1) and rat (A2-2 and A3-3) cDNA sequences was constructed by the method of DNA shuffling. The GST variants were screened in bacterial lysates for activity with the immunosuppressive agent azathioprine, a prodrug that is transformed into its active form, 6-mercaptopurine, by reaction with the tripeptide glutathione catalyzed by GSTs. Important structural determinants for activity with azathioprine were recognized by means of primary structure analysis and activities of purified enzymes chosen from the screening. The amino acid sequences could be divided into 23 exchangeable segments on the basis of the primary structures of 45 chosen clones. Segments 2, 20, 21, and 22 were identified as primary determinants of the azathioprine activity representing two of the regions forming the substrate-binding H-site. Segments 21 and 22 are situated in the C-terminal helix characterizing alpha class GSTs, which is instrumental in their catalytic function. The study demonstrates the power of DNA shuffling in identifying segments of primary structure that are important for catalytic activity with a targeted substrate. GSTs in combination with azathioprine have potential as selectable markers for use in gene therapy. Knowledge of activity-determining segments in the structure is valuable in the protein engineering of glutathione transferase for enhanced or suppressed activity.
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| 3. |
- Modén, Olof
(författare)
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Mutational Analysis and Redesign of Alpha-class Glutathione Transferases for Enhanced Azathioprine Activity
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glutathione transferase (GST) A2-2 is the human enzyme most efficient in catalyzing azathioprine activation. Structure-function relationships were sought explaining the higher catalytic efficiency compared to other alpha class GSTs. By screening a DNA shuffling library, five recombined segments were identified that were conserved among the most active mutants. Mutational analysis confirmed the importance of these short segments as their insertion into low-active GSTs introduced higher azathioprine activity. Besides, H-site mutagenesis led to decreased azathioprine activity when the targeted positions belonged to these conserved segments and mainly enhanced activity when other positions were targeted. Hydrophobic residues were preferred in positions 208 and 213.The prodrug azathioprine is today primarily used for maintaining remission in inflammatory bowel disease. Therapy leads to adverse effects for 30 % of the patients and genotyping of the metabolic genes involved can explain some of these incidences. Five genotypes of human A2-2 were characterized and variant A2*E had 3–4-fold higher catalytic efficiency with azathioprine, due to a proline mutated close to the H-site. Faster activation might lead to different metabolite distributions and possibly more adverse effects. Genotyping of GSTs is recommended for further studies.Molecular docking of azathioprine into a modeled structure of A2*E suggested three positions for mutagenesis. The most active mutants had small or polar residues in the mutated positions. Mutant L107G/L108D/F222H displayed a 70-fold improved catalytic efficiency with azathioprine. Determination of its structure by X-ray crystallography showed a widened H-site, suggesting that the transition state could be accommodated in a mode better suited for catalysis.The mutational analysis increased our understanding of the azathioprine activation in alpha class GSTs and highlighted A2*E as one factor possibly behind the adverse drug-effects. A successfully redesigned GST, with 200-fold enhanced catalytic efficiency towards azathioprine compared to the starting point A2*C, might find use in targeted enzyme-prodrug therapies.
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| 4. |
- Modén, Olof, et al.
(författare)
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Mutational analysis of human glutathione transferase A2-2 identifies structural elements supporting high activity with the prodrug azathioprine
- 2012
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 25:4, s. 189-197
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferase (GST) A2-2 is the human enzyme displaying the highest catalytic activity with the prodrug azathioprine (Aza). The reaction releases pharmacologically active 6-mercaptopurine by displacing the imidazole moiety from the Aza molecule. The GST-catalyzed reaction is of medical significance, since high rates of Aza activation may lead to adverse side effects in treated patients. The present study involves structureactivity relationships in GST A2-2 variants. Chimeric GSTs were previously generated by DNA shuffling and two peptide segments, one N-terminal and one C-terminal, were identified as primary determinants of Aza activity. The segments contain several residues of the substrate-binding H-site and their significance for supporting high Aza activity was investigated. Substitution of the corresponding two small regions in the low-activity human GST A3-3 or rat GST A3-3 by the human GST A2-2 segments generated chimeras with approximate to 10-fold enhanced Aza activity. The H-site residues Met208 and Leu213 in the C-terminal segment of GST A2-2 were mutated to produce a library with all possible residue combinations. At a calculated 93 library coverage, all of the 1880 mutants examined showed wild-type or decreased Aza activity, even though some retained activities with alternative substrates, further emphasizing the importance of this region for the targeted activity.
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| 5. |
- Zhang, Wei, et al.
(författare)
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Differences among allelic variants of human glutathione transferase A2-2 in the activation of azathioprine
- 2010
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Ingår i: Chemico-Biological Interactions. - : Elsevier BV. - 0009-2797 .- 1872-7786. ; 186:2, s. 110-117
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Tidskriftsartikel (refereegranskat)abstract
- Azathioprine has been clinically used for decades in connection with organ transplantation, autoimmune disease, and treatment of cancer. Toxic side-reactions are common and have been linked to the liberation of excessively high concentrations of 6-mercaptopurine and corresponding toxic metabolites. An allelic variant of thiopurine methyltransferase with low activity is associated with elevated concentrations of 6-mercaptopurine. However, other genetic markers remain to be identified in order to fully account for adverse reactions and efficacy failure. In the present study, we studied the five known allelic variants of human glutathione transferase A2-2 (GST A2-2) (EC2.5.1.18), abundantly expressed in liver and efficiently catalyzing the bioactivation of azathioprine to release 6-mercaptopurine. All five variants exhibited high activity with azathioprine, but allelic variant E of GST A2-2 displayed a 3-4-fold elevated catalytic efficiency compared to the other variants. High GST activity can lead to overproduction of 6-mercaptopurine, and the nature of the multiple forms of GSTs in a patient will obviously affect the metabolism of azathioprine. In addition to GST A2-2, the polymorphic GST M1-1 is also highly active with azathioprine. Considering our findings, it appears that the genotypic and phenotypic variations in the GST complement may influence the presentation of adverse reactions in patients treated with azathioprine. Clinical trials will be required to clarify the impact of the GST expression in comparison with the established biomarker thiopurine methyltransferase as predictors of adverse reactions.
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| 6. |
- Zhang, Wei, et al.
(författare)
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Structure-based redesign of GST A2-2 for enhanced catalytic efficiency with azathioprine
- 2012
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 19:3, s. 414-421
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferase (GST) A2-2 is the most efficient human enzyme in the biotransformation of the prodrug azathioprine (Aza). The activation of Aza has therapeutic potential for possible use of GSTs in targeted enzyme-prodrug treatment of diseases. Based on the assumed catalytic mechanism and computational docking of Aza to the active site of the enzyme, active-site residues were selected for construction of focused mutant libraries, which were thereafter screened for Aza activity. Mutants with elevated Aza activity were identified, DNA sequenced, and the proteins purified. The two most active mutants showed up to 70-fold higher catalytic efficiency than the parental GST A2-2. The structure of the most active triple mutant (L107G/L108D/F222H) enzyme was determined by X-ray crystallography demonstrating significant changes in the topography of the active site facilitating productive binding of Aza as a substrate.
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