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Structural determinants of glutathione transferases with azathioprine activity identified by DNA shuffling of alpha class members
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.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 375, no 5, 1365-1379 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2008. Vol. 375, no 5, 1365-1379 p.
Keyword [en]
Azathioprine, DNA shuffling, glutathione transferase, protein modification, rational redesign, directed evolution, drug resistance, prodrug, SAR
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Identifiers
URN: urn:nbn:se:uu:diva-97180DOI: 10.1016/j.jmb.2007.11.034ISI: 000253098100016PubMedID: 18155239OAI: oai:DiVA.org:uu-97180DiVA: diva2:172003
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Directed Evolution of Glutathione Transferases Guided by Multivariate Data Analysis
Open this publication in new window or tab >>Directed Evolution of Glutathione Transferases Guided by Multivariate Data Analysis
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Evolution of enzymes with novel functional properties has gained much attention in recent years. Naturally evolved enzymes are adapted to work in living cells under physiological conditions, circumstances that are not always available for industrial processes calling for novel and better catalysts. Furthermore, altering enzyme function also affords insight into how enzymes work and how natural evolution operates.

Previous investigations have explored catalytic properties in the directed evolution of mutant libraries with high sequence variation. Before this study was initiated, functional analysis of mutant libraries was, to a large extent, restricted to uni- or bivariate methods. Consequently, there was a need to apply multivariate data analysis (MVA) techniques in this context. Directed evolution was approached by DNA shuffling of glutathione transferases (GSTs) in this thesis. GSTs are multifarious enzymes that have detoxication of both exo- and endogenous compounds as their primary function. They catalyze the nucleophilic attack by the tripeptide glutathione on many different electrophilic substrates.

Several multivariate analysis tools, e.g. principal component (PC), hierarchical cluster, and K-means cluster analyses, were applied to large mutant libraries assayed with a battery of GST substrates. By this approach, evolvable units (quasi-species) fit for further evolution were identified. It was clear that different substrates undergoing different kinds of chemical transformation can group together in a multi-dimensional substrate-activity space, thus being responsible for a certain quasi-species cluster. Furthermore, the importance of the chemical environment, or substrate matrix, in enzyme evolution was recognized. Diverging substrate selectivity profiles among homologous enzymes acting on substrates performing the same kind of chemistry were identified by MVA. Important structure-function activity relationships with the prodrug azathioprine were elucidated by segment analysis of a shuffled GST mutant library. Together, these results illustrate important methods applied to molecular enzyme evolution.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 82 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 431
Keyword
Biochemistry, DNA shuffling, substrate selectivity, mutant library, glutathione transferase, multivariate data analysis, prodrug, Biokemi
Identifiers
urn:nbn:se:uu:diva-8718 (URN)978-91-554-7194-1 (ISBN)
Public defence
2008-05-23, B7:101a, BMC, Box 576, Uppsala University, SE-75123 Uppsala, 09:15
Opponent
Supervisors
Available from: 2008-04-29 Created: 2008-04-29Bibliographically 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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