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The Quest for Functional Quasi-Species in Glutathione Transferase Libraries
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
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. , p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 737
Keywords [en]
glutathione transferase, directed evolution, multivariate analysis, quasi-species, isothiocyanates
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-122378ISBN: 978-91-554-7794-3 (print)OAI: oai:DiVA.org:uu-122378DiVA, id: diva2:309943
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
List of papers
1. Multivariate-activity mining for molecular quasi-species in a glutathione transferase mutant library
Open this publication in new window or tab >>Multivariate-activity mining for molecular quasi-species in a glutathione transferase mutant library
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2007 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 5, p. 243-256Article 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.

Keywords
directed evolution, DNA shuffling, glutathione transferase, library, multivariate analysis
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-97179 (URN)10.1093/protein/gzm017 (DOI)000247313600006 ()17468114 (PubMedID)
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
2. Functionally diverging molecular quasi-species evolve by crossing two enzymes
Open this publication in new window or tab >>Functionally diverging molecular quasi-species evolve by crossing two enzymes
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2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 29, p. 10866-10870Article in journal (Refereed) Published
Abstract [en]

Molecular evolution is frequently portrayed by structural relationships, but delineation of separate functional species is more elusive. We have generated enzyme variants by stochastic recombinations of DNA encoding two homologous detoxication enzymes, human glutathione transferases M1-1 and M2-2, and explored their catalytic versatilities. Sampled mutants were screened for activities with eight alternative substrates, and the activity fingerprints were subjected to principal component analysis. This phenotype characterization clearly identified at least three distributions of substrate selectivity, where one was orthogonal to those of the parent-like distributions. This approach to evolutionary data mining serves to identify emerging molecular quasi-species and indicates potential trajectories available for further protein evolution.

Keywords
directed evolution, DNA shuffling, glutathione transferase, library, multivariate analysis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-97178 (URN)10.1073/pnas.0604030103 (DOI)000239327200010 ()16829572 (PubMedID)
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
3. Multi-substrate-activity space and quasi-species in enzyme evolution: Ohno's dilemma, promiscuity and functional orthogonality
Open this publication in new window or tab >>Multi-substrate-activity space and quasi-species in enzyme evolution: Ohno's dilemma, promiscuity and functional orthogonality
2009 (English)In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 37, no Pt 4, p. 740-744Article 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.

Keywords
directed evolution, enzyme activity space, enzyme promiscuity, functional orthogonality, glutathione transferases (GST), quasi-species
National Category
Other Clinical Medicine
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-122374 (URN)10.1042/BST0370740 (DOI)000268902800020 ()19614586 (PubMedID)
Available from: 2010-04-09 Created: 2010-04-09 Last updated: 2017-12-12Bibliographically approved
4. A novel quasi-species of glutathione transferase with high activity towards naturally occurring isothiocyanates evolves from promiscuous low-activity variants
Open this publication in new window or tab >>A novel quasi-species of glutathione transferase with high activity towards naturally occurring isothiocyanates evolves from promiscuous low-activity variants
2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 401, no 3, p. 451-464Article 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.

Keywords
functional quasi-species, glutathione transferase, substrate matrix, multivariate analysis, isothiocyanates, activity profiles
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-122377 (URN)10.1016/j.jmb.2010.06.033 (DOI)000281171000010 ()20600121 (PubMedID)
Note
Uppdaterad från Manuskript till Artikel 20101203Available from: 2010-04-09 Created: 2010-04-09 Last updated: 2017-12-12Bibliographically approved
5. The quest for molecular quasi-species in ligand-activity space and its application to directed enzyme evolution
Open this publication in new window or tab >>The quest for molecular quasi-species in ligand-activity space and its application to directed enzyme evolution
2010 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 584, no 12, p. 2565-2571Article, review/survey (Refereed) Published
Abstract [en]

We propose that the proper evolving unit in enzyme evolution is not a single "fittestmolecule", but a cluster of related variants denoted a "quasi-species". A distribution of variantsprovides genetic variability and thereby reduces the risk of inbreeding and evolutionary dead-ends.Different matrices of substrates or activity modulators will lead to different selection criteria anddivergent evolutionary trajectories. We provide examples from our directed evolution of glutathionetransferases illustrating the interplay between libraries of enzyme variants and ligand matrices in theidentification of quasi-species. The ligand matrix is shown to be crucial to the outcome of the search fornovel activities. In this sense the experimental system resembles the biological environment ingoverning the evolution of enzymes.

Keywords
quasi-species, glutathione transferase, enzyme evolution, ligand space, activity space, multivariate analysis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-122376 (URN)10.1016/j.febslet.2010.04.024 (DOI)000278031800010 ()
Available from: 2010-04-09 Created: 2010-04-09 Last updated: 2017-12-12Bibliographically approved

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