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Emergence of novel enzyme quasi-species depends on the substrate matrix
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Biokemi I)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Biokemi I)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Biokemi I)
2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 382, no 1, 136-153 p.Article in journal (Refereed) Published
Abstract [en]

Current research on enzyme evolution has shown that many enzymes are promiscuous and have activities with alternative substrates. Mutagenesis tends to relax substrate selectivity, and evolving enzymes can be regarded" quasi(summed over evolutionary time) as clusters of enzyme variants, or species," tested against a "substrate matrix" defined by all chemical substances to which the evolvants are exposed.In this investigation, the importance of the substrate matrix for identification of evolvable clusters of enzymes was evaluated by random sampling of variants from a library of glutathione transferase (GST) mutants. The variant GSTs were created by DNA shuffling of homologous Alpha class sequences. The substrate matrix was an array of alternative substrates used under defined experimental conditions. The measured enzyme activities produced a rectangular matrix, in which the rows can be projected as enzyme vectors in substrate-activity space and, reciprocally, the columns can be projected as alternative substrate vectors in enzyme-activity space. Multivariate analysis of the catalytic activities demonstrated that the enzyme vectors formed two primary clusters or functional "molecular quasi-species." These quasi-species serve as the raw material from which more specialized enzymes eventually could evolve. The substrate vectors similarly formed two major groups. Identification of separate quasi-species of GSTs in a mutant library was critically dependent on the nature of the substrate matrix. When substrates from just one of the two groups were used, only one cluster of enzymes could be recognized. On the other hand, expansion of the substrate matrix to include additional substrates showed the presence of a third quasi-species among the GST variants already analyzed. Thus, the portrayal of the functional quasi-species is intimately linked to the effective substrate matrix. In natural evolution, the substrates actually encountered therefore play a pivotal role in determining whether latent catalytic abilities become manifest in novel enzymes.

Place, publisher, year, edition, pages
2008. Vol. 382, no 1, 136-153 p.
Keyword [en]
DNA shuffling, glutathione transferase, multivariate data analysis
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-97184DOI: 10.1016/j.jmb.2008.07.003ISI: 000259588500012PubMedID: 18640124OAI: oai:DiVA.org:uu-97184DiVA: diva2:172007
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2009-08-28Bibliographically 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

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