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Diverging catalytic capacities and selectivity profiles with haloalkane substrates of chimeric alpha class glutathione transferases
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: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, no 5, 329-341 p.Article in journal (Refereed) Published
Abstract [en]

Six homologous Alpha class glutathione transferases of human, bovine, and rat origins were hybridized by means of DNA shuffling. The chimeric mutants were compared with the parental enzymes in their activities with several alkyl iodides. In order to facilitate a multivariate analysis of relationships between substrates and enzyme activities, three descriptors were introduced: 'specific catalytic capacity', 'substrate selectivity', and 'unit-scaled substrate selectivity'. In some cases the purified mutants showed higher specific activity with a certain alkyl iodide than any of the parental enzymes. However, the overriding effect of DNA shuffling was the generation of chimeras with altered substrate selectivity profiles and catalytic capacities. The altered substrate selectivity profiles of some mutants could be rationalized by changes of the substrate-binding residues in the active site of the enzyme. However, in four of the isolated mutants all active-site residues were found identical with those of rat GST A2-2, even though their substrate specificity profiles were significantly different. Clearly, amino acid residues distant from first-sphere interactions with the substrate influence the catalytic activity. These results are relevant both to the understanding how functional properties may develop in natural enzyme evolution and in the tailoring of novel functions in protein engineering.

Place, publisher, year, edition, pages
2008. Vol. 21, no 5, 329-341 p.
Keyword [en]
DNA shuffling, glutathione transferase, multivariate data analysis, mutant library, substrate selectivity
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-97183DOI: 10.1093/protein/gzn010ISI: 000255156400007OAI: oai:DiVA.org:uu-97183DiVA: diva2:172006
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

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