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Mutational analysis of human glutathione transferase A2-2 identifies structural elements supporting high activity with the prodrug azathioprine
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
2012 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 25, no 4, 189-197 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2012. Vol. 25, no 4, 189-197 p.
Keyword [en]
directed evolution, DNA shuffling, GST, prodrug, structure-activity relationship, azathioprine, chimeric mutagenesis, glutathione transferase, prodrug activation, saturation mutagenesis
National Category
Biochemistry and Molecular Biology Biocatalysis and Enzyme Technology
Identifiers
URN: urn:nbn:se:uu:diva-167324DOI: 10.1093/protein/gzs006ISI: 000302021100006OAI: oai:DiVA.org:uu-167324DiVA: diva2:483596
Available from: 2012-01-25 Created: 2012-01-25 Last updated: 2017-12-08Bibliographically approved
In thesis
1. 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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Modén, OlofZhang, WeiMannervik, Bengt

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