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

Direct link
A novel quasi-species of glutathione transferase with high activity towards naturally occurring isothiocyanates evolves from promiscuous low-activity variants
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.
2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 401, no 3, 451-464 p.Article in journal (Refereed) Published
Abstract [en]

Glutathione transferases (GSTs) are known as promiscuous enzymes capable of catalyzing the conjugation of glutathione with a broad range of electrophilic substrates. A previous study based on recombinant chimeras derived from human GST M1-1 and GST M2-2 demonstrated the formation of a subset of F1-generation GSTs, which had lost the high activity for substrates distinguishing the parental enzymes. In the present study the members of this subset were recombined by DNA shuffling to produce an F2 generation of GSTs. Screening of 930 bacterial clones demonstrated that 83% of the recombinant enzyme variants were active with at least one of three alternative substrates: phenethyl isothiocyanate (PEITC), 1-chloro-2,4-dinitrobenzene (CDNB), or p-nitrophenyl acetate (pNPA). The majority had similar low activity as the parental GSTs in the F1 generation. However, 17 novel enzymes displayed high activity with PEITC. Half of them were similar to GST M1-1, which also has high activity with the same substrate, and all of these GSTs featured Tyr116/Ser210 in the active site. This group of F2 variants apparently had reverted to the GST M1-1 type. A second group of F2 variants with high PEITC activity was characterized by His116 in the active site. This category represented a new variety of GSTs, which demonstrated higher selectivity for isothiocyanate substrates than the GST M1-1 type. The different groups of GSTs can be considered as distinct molecular quasi-species each of which comprising variant amino acid sequences. The quasi-species are structurally distinguished by the active-site residues that govern their substrate selectivities. Clearly, minimal alterations of the active site can generate enzymes with highly distinctive functional properties.

Place, publisher, year, edition, pages
2010. Vol. 401, no 3, 451-464 p.
Keyword [en]
functional quasi-species, glutathione transferase, substrate matrix, multivariate analysis, isothiocyanates, activity profiles
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-122377DOI: 10.1016/j.jmb.2010.06.033ISI: 000281171000010PubMedID: 20600121OAI: oai:DiVA.org:uu-122377DiVA: diva2:309931
Uppdaterad från Manuskript till Artikel 20101203Available from: 2010-04-09 Created: 2010-04-09 Last updated: 2010-12-03Bibliographically 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 737
glutathione transferase, directed evolution, multivariate analysis, quasi-species, isothiocyanates
Research subject
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)
Available from: 2010-04-28 Created: 2010-04-09 Last updated: 2011-06-30Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Runarsdottir, Arna
By organisation
Department of Biochemistry and Organic Chemistry
In the same journal
Journal of Molecular Biology
Biological 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: 152 hits
ReferencesLink to record
Permanent link

Direct link