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Multi-substrate-activity space and quasi-species in enzyme evolution: Ohno's dilemma, promiscuity and functional orthogonality
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.
2009 (English)In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 37, no Pt 4, 740-744 p.Article in journal (Refereed) Published
Abstract [en]

A functional enzyme displays activity with at least one substrate and can be represented by a vector in substrate-activity space. Many enzymes, including GSTs (glutathione transferases), are promiscuous in the sense that they act on alternative substrates, and the corresponding vectors operate in multidimensional space. The direction of the vector is governed by the relative activities of the diverse substrates. Stochastic mutations of already existing enzymes generate populations of variants, and clusters of functionally similar mutants can serve as parents for subsequent generations of enzymes. The proper evolving unit is a functional quasi-species, which may not be identical with the 'best' variant in its generation. The manifestation of the quasi-species is dependent on the substrate matrix used to explore catalytic activities. Multivariate analysis is an approach to identifying quasi-species and to investigate evolutionary trajectories in the directed evolution of enzymes for novel functions.

Place, publisher, year, edition, pages
2009. Vol. 37, no Pt 4, 740-744 p.
Keyword [en]
directed evolution, enzyme activity space, enzyme promiscuity, functional orthogonality, glutathione transferases (GST), quasi-species
National Category
Other Clinical Medicine
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-122374DOI: 10.1042/BST0370740ISI: 000268902800020PubMedID: 19614586OAI: oai:DiVA.org:uu-122374DiVA: diva2:309929
Available from: 2010-04-09 Created: 2010-04-09 Last updated: 2011-03-11Bibliographically approved
In thesis
1. 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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