uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Glutathione transferase activity with a novel substrate mimics the activation of the prodrug azathioprine
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Medicago AB, Danmark Berga, Uppsala SE-75598, Sweden.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
Show others and affiliations
2008 (English)In: Analytical Biochemistry, ISSN 0003-2697, Vol. 375, no 2, 339-344 p.Article in journal (Refereed) Published
Abstract [en]

Azathioprine is a prodrug that is widely used clinically as an immunosuppressive agent. The pharmacological action of azathioprine is associated with the release of 6-mercaptopurine by a reaction involving glutathione. This biotransformation of azathioprine is catalyzed by glutathione transferases (GSTs). The nonenzymatic reaction with glutathione is minimal in comparison with the GST-catalyzed process, but azathioprine is still a slow substrate in comparison with the most effective GST substrates. Novel GSTs with higher catalytic efficiency toward azathioprine could be useful in novel therapeutic applications; therefore, directed evolution of GSTs for enhanced activities is desirable. However, screening for variants having higher catalytic activity with azathioprine is a time-consuming process due to the low activity with this substrate. A new chromogenic and faster substrate, 1-methyl-4-nitro-5-(4-nitrophenylthio)-1H-imidazole (NPTI), has been synthesized and characterized by assays with several GSTs. The novel substrate mimicked azathioprine in the reaction with glutathione catalyzed by alpha class GSTs and, therefore, is a valuable surrogate in the screening of large mutant libraries. NPTI may also find use in the elucidation of the exact mechanism of immunosuppression effected by azathioprine where there is evidence that the imidazole moiety of azathioprine, rather than 6-mercaptopurine, is involved.

Place, publisher, year, edition, pages
2008. Vol. 375, no 2, 339-344 p.
Keyword [en]
Azathioprine, Enzyme assay, GST, Prodrug mimetic
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-97181DOI: 10.1016/j.ab.2007.12.033ISI: 000254410800022PubMedID: 18211812OAI: oai:DiVA.org:uu-97181DiVA: diva2:172004
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2009-11-12Bibliographically 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 431
Biochemistry, DNA shuffling, substrate selectivity, mutant library, glutathione transferase, multivariate data analysis, prodrug, Biokemi
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
Available from: 2008-04-29 Created: 2008-04-29Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed
By organisation
Department of Biochemistry and Organic ChemistryLudwig Institute for Cancer Research
In the same journal
Analytical Biochemistry
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 209 hits
ReferencesLink to record
Permanent link

Direct link