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Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles: A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
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 [en]
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: urn:nbn:se:uu:diva-158400ISBN: 978-91-554-8147-6 (print)OAI: oai:DiVA.org:uu-158400DiVA: diva2:439229
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
List of papers
1. Differences among allelic variants of human glutathione transferase A2-2 in the activation of azathioprine
Open this publication in new window or tab >>Differences among allelic variants of human glutathione transferase A2-2 in the activation of azathioprine
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.

Keyword
Azathioprine, Glutathione transferase, Bioactivation, Thiopurine methyltransferase, Allelic variants, Polymorphism
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:uu:diva-135839 (URN)10.1016/j.cbi.2010.04.028 (DOI)000279644900002 ()
Available from: 2010-12-08 Created: 2010-12-08 Last updated: 2017-12-11Bibliographically approved
2. Structure-based redesign of GST A2-2 for enhanced catalytic efficiency with azathioprine
Open this publication in new window or tab >>Structure-based redesign of GST A2-2 for enhanced catalytic efficiency with azathioprine
2012 (English)In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 19, no 3, 414-421 p.Article in journal (Refereed) Published
Abstract [en]

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. 

Keyword
glutathione transferase, azathioprine, structure-based redesign, semi-rational enzyme engineering, reduced amino acid alphabet, directed evolution
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:uu:diva-158395 (URN)10.1016/j.chembiol.2012.01.021 (DOI)000302588900014 ()
Available from: 2011-09-06 Created: 2011-09-06 Last updated: 2017-12-08Bibliographically approved
3. Multidimensional epistasis and fitness landscapes in enzyme evolution
Open this publication in new window or tab >>Multidimensional epistasis and fitness landscapes in enzyme evolution
Show others...
2012 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 445, 39-46 p.Article 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.

Keyword
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
4. An improved dual-tube megaprimer approach for multi-site saturation mutagenesis
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, 667-672 p.Article 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

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