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Differences among allelic variants of human glutathione transferase A2-2 in the activation of azathioprine
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2010 (English)In: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 186, no 2, 110-117 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2010. Vol. 186, no 2, 110-117 p.
Keyword [en]
Azathioprine, Glutathione transferase, Bioactivation, Thiopurine methyltransferase, Allelic variants, Polymorphism
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Identifiers
URN: urn:nbn:se:uu:diva-135839DOI: 10.1016/j.cbi.2010.04.028ISI: 000279644900002OAI: oai:DiVA.org:uu-135839DiVA: diva2:375720
Available from: 2010-12-08 Created: 2010-12-08 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles: A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis
Open this publication in new window or tab >>Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles: A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Directed evolution is generally regarded as a useful approach in protein engineering. By subjecting members of a mutant library to the power of Darwinian evolution, desired protein properties are obtained. Numerous reports have appeared in the literature showing the success of tailoring proteins for various applications by this method. Is it a one-way track that protein practitioners can only learn from nature to enable more efficient protein engineering?

A structure-and-mechanism-based approach, supplemented with the use of reduced amino acid alphabets, was proposed as a general means for semi-rational enzyme engineering. Using human GST A2-2*E, the most active human enzyme in the bioactivation of azathioprine, as a parental enzyme to test this approach, a L107G/L108D/F222H triple-point mutant of GST A2-2*E (thereafter designated as GDH) was discovered with 70-fold increased activity, approaching the upper limit of specific activity of the GST scaffold. The approach was further experimentally verified to be more successful than intuitively choosing active-site residues in proximity to the bound substrate for the improvement of enzyme performance.

By constructing all intermediates along all putative mutational paths leading from GST A2-2*E to mutant GDH and assaying them with nine alternative substrates, the fitness landscapes were found to be “rugged” in differential fashions in substrate-activity space. The multidimensional fitness landscapes stemming from functional promiscuity can lead to alternative outcomes with enzymes optimized for other features than the selectable markers that were relevant at the origin of the evolutionary process. The results in this thesis suggest that in this manner an evolutionary response to changing environmental conditions can readily be mounted.

In summary, the thesis demonstrates the attractive features of the structure-and-mechanism-based semi-rational directed evolution approach for optimizing enzyme performance. Moreover, the results gained from the studies show that laboratory evolution may refine our understanding of evolutionary process in nature.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 47 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 850
Keyword
glutathione transferase, azathioprine, directed evolution, semi-rational design, catalytic mechanism, saturation mutagenesis, reduced amino acid alphabets, molecular docking, protein evolution, multivariate data analysis, epistasis, fitness landscape, evolutionary trajectories
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-158400 (URN)978-91-554-8147-6 (ISBN)
Public defence
2011-10-21, C2:301, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2011-09-30 Created: 2011-09-06 Last updated: 2011-11-03Bibliographically approved
2. Mutational Analysis and Redesign of Alpha-class Glutathione Transferases for Enhanced Azathioprine Activity
Open this publication in new window or tab >>Mutational Analysis and Redesign of Alpha-class Glutathione Transferases for Enhanced Azathioprine Activity
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1050
Keyword
allelic variants, azathioprine, bioactivation, chimeric mutagenesis, directed evolution, DNA shuffling, enzyme engineering, glutathione transferase, GST, lysate screening, molecular docking, multiple alignment, multivariate analysis, polymorphism, principal component analysis, prodrug, prodrug activation, protein engineering, protein redesign, reduced amino acid alphabet, saturation mutagenesis, semi-rational enzyme engineering, site-directed mutagenesis, structure-activity relationship, structure-based redesign
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-167332 (URN)978-91-554-8685-3 (ISBN)
Public defence
2013-06-05, B42, Biomedical Center (BMC), Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2013-05-15 Created: 2012-01-25 Last updated: 2013-08-30Bibliographically approved

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Zhang, WeiModén, Olof

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