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Multidimensional epistasis and fitness landscapes in enzyme evolution
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Universidade do Porto Rua do Campo Alegre.
Universidade do Porto Rua do Campo Alegre.
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2012 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 445, 39-46 p.Article in journal (Refereed) Published
Abstract [en]

The conventional analysis of enzyme evolution is to regard one single salient feature as a measure of fitness, expressed in a milieu exposing the possible selective advantage at a given time and location. Given that a single protein may serve more than one function, fitness should be assessed in several dimensions. In the present study we have explored individual mutational steps leading to a triple-point-mutated human GST (glutathione transferase) A2-2 displaying enhanced activity with azathioprine. A total of eight alternative substrates were used to monitor the diverse evolutionary trajectories. The epistatic effects of the imitations on catalytic activity were variable in sign and magnitude and depended on the substrate used, showing that epistasis is a multidimensional quality. Evidently, the multidimensional fitness landscape can lead to alternative trajectories resulting in enzymes optimized for features other than the selectable markers relevant at the origin of the evolutionary process. In this manner the evolutionary response is robust and can adapt to changing environmental conditions.

Place, publisher, year, edition, pages
2012. Vol. 445, 39-46 p.
Keyword [en]
epistasis, evolutionary trajectories, fitness landscape, multivariate data analysis, protein evolution, substrate selectivity
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-158397DOI: 10.1042/BJ20120136ISI: 000306874300004OAI: oai:DiVA.org:uu-158397DiVA: diva2:439218
Note

Original manuscript title: "Epistasis is a multidimensional property in the evolutionary trajectories of glutathione transferase in alternative-substrate-activity space"

Available from: 2011-09-06 Created: 2011-09-06 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles: A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis
Open this publication in new window or tab >>Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles: A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Directed evolution is generally regarded as a useful approach in protein engineering. By subjecting members of a mutant library to the power of Darwinian evolution, desired protein properties are obtained. Numerous reports have appeared in the literature showing the success of tailoring proteins for various applications by this method. Is it a one-way track that protein practitioners can only learn from nature to enable more efficient protein engineering?

A structure-and-mechanism-based approach, supplemented with the use of reduced amino acid alphabets, was proposed as a general means for semi-rational enzyme engineering. Using human GST A2-2*E, the most active human enzyme in the bioactivation of azathioprine, as a parental enzyme to test this approach, a L107G/L108D/F222H triple-point mutant of GST A2-2*E (thereafter designated as GDH) was discovered with 70-fold increased activity, approaching the upper limit of specific activity of the GST scaffold. The approach was further experimentally verified to be more successful than intuitively choosing active-site residues in proximity to the bound substrate for the improvement of enzyme performance.

By constructing all intermediates along all putative mutational paths leading from GST A2-2*E to mutant GDH and assaying them with nine alternative substrates, the fitness landscapes were found to be “rugged” in differential fashions in substrate-activity space. The multidimensional fitness landscapes stemming from functional promiscuity can lead to alternative outcomes with enzymes optimized for other features than the selectable markers that were relevant at the origin of the evolutionary process. The results in this thesis suggest that in this manner an evolutionary response to changing environmental conditions can readily be mounted.

In summary, the thesis demonstrates the attractive features of the structure-and-mechanism-based semi-rational directed evolution approach for optimizing enzyme performance. Moreover, the results gained from the studies show that laboratory evolution may refine our understanding of evolutionary process in nature.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 47 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 850
Keyword
glutathione transferase, azathioprine, directed evolution, semi-rational design, catalytic mechanism, saturation mutagenesis, reduced amino acid alphabets, molecular docking, protein evolution, multivariate data analysis, epistasis, fitness landscape, evolutionary trajectories
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-158400 (URN)978-91-554-8147-6 (ISBN)
Public defence
2011-10-21, C2:301, BMC, Husargatan 3, Uppsala, 10:15 (English)
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Supervisors
Available from: 2011-09-30 Created: 2011-09-06 Last updated: 2011-11-03Bibliographically approved

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Zhang, WeiDourado, Daniel F. A. R.Mannervik, Bengt

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