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Multivariate-activity mining for molecular quasi-species in a glutathione transferase mutant library
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.
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2007 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 5, 243-256 p.Article in journal (Refereed) Published
Abstract [en]

A library of recombinant glutathione transferases (GSTs) generated by shuffling of DNA encoding human GST M1-1 and GST M2-2 was screened with eight alternative substrates, and the activities were subjected to multivariate analysis. Assays were made in lysates of bacteria in which the GST variants had been expressed. The primary data showed clustering of the activities in eight-dimensional substrate-activity space. For an incisive analysis, the rows of the data matrix, corresponding to the different enzyme variants, were individually scaled to unit length, thus accounting for different expression levels of the enzymes. The columns representing the activities with alternative substrates were subsequently individually normalized to unit variance and a zero mean. By this standardization, the data were adjusted to comparable orders of magnitude. Three molecular quasi-species were recognized by multivariate K-means and principal component analyses. Two of them encompassed the parental GST M1-1 and GST M2-2. A third one diverged functionally by displaying enhanced activities with some substrates and suppressed activities with signature substrates for GST M1-1 and GST M2-2. A fourth cluster contained mutants with impaired functions and was not regarded as a quasi-species. Sequence analysis of representatives of the mutant clusters demonstrated that the majority of the variants in the diverging novel quasi-species were structurally similar to the M1-like GSTs, but distinguished themselves from GST M1-1 by a Ser to Thr substitution in the active site. The data show that multivariate analysis of functional profiles can identify small structural changes influencing the evolution of enzymes with novel substrate-activity profiles.

Place, publisher, year, edition, pages
2007. Vol. 20, no 5, 243-256 p.
Keyword [en]
directed evolution, DNA shuffling, glutathione transferase, library, multivariate analysis
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97179DOI: 10.1093/protein/gzm017ISI: 000247313600006PubMedID: 17468114OAI: oai:DiVA.org:uu-97179DiVA: diva2:172002
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. The Quest for Functional Quasi-Species in Glutathione Transferase Libraries
Open this publication in new window or tab >>The Quest for Functional Quasi-Species in Glutathione Transferase Libraries
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs) are good candidates for investigations of enzyme evolution, due to their broad substrate specificities and structural homology. The primary role of GSTs is to act as phase II detoxifying enzymes protecting the cell from toxic compounds of both endo- and exogenous origins. The detoxification is conducted via conjugation with glutathione (GSH), which facilitates their removal from the body.

The work presented in this thesis has supported a theory for enzyme evolution when the multiple pathway to novel functions can been seen to involve a “generalist” state from which “specialist” states with a new activities can evolve. The generalist has broader specificity and lower activity than the specialist. The term quasi-species is used for a group or cluster of enzyme variants with similar functional properties, and this entity has been suggested as the fittest group for further evolution. This is based on studies of the evolution of new GST variants in two generation.

Three diverging clusters or quasi-species, with diverging substrate selectivity, were identified from a GST M1/M2 library, by using directed evolution (family DNA shuffling), multiple substrate screening and multivariate statistics as tools. One of the clusters was M1-like and the other was M2-like, both functionally and structurally. The third quasi-species diverged orthogonally from the parent-like distributions. Its functional character can be referred to as a “generalist” as it had lower activities with most of the substrates assayed except for epoxy-3-(4-nitrophenoxy)-propane (EPNP) and p-nitrophenyl acetate (pNPA).

Another round of family DNA shuffling was made with selected variants from the “generalist” quasi-species. From the second generation three quasi-species emerged with diverging functions and sequences. The major cluster contained enzyme variants that represented a direct propagation of the generalists. Diverging from the generalists was a cluster with high specificity with isothiocyanates (ITCs). Increased ITC specificity and decreased epoxide specificity was observed among the novel variants (specialists). The change in functional properties was attributed to a Tyr116His substitution in the active site.

These results demonstrate the usefulness of multivariate analysis in the quest for novel enzyme quasi-species in a multi-substrate space, and how minimal changes in the active site can generate distinctive functional properties. An application of our method could be identification of enzyme quasi-species that have lost their sensitivity with alternative inhibitors.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 60 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 737
Keyword
glutathione transferase, directed evolution, multivariate analysis, quasi-species, isothiocyanates
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-122378 (URN)978-91-554-7794-3 (ISBN)
Public defence
2010-05-19, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2010-04-28 Created: 2010-04-09 Last updated: 2011-06-30Bibliographically approved

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Kurtovic, SanelaRunarsdottir, ArnaMannervik, Bengt

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