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Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications
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 Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2007 (English)In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1770, no 9, 1374-1381 p.Article in journal (Refereed) Published
Abstract [en]

Based on the crystal structure of human glutathione transferase M1-1, cysteine residues were introduced in the substrate-binding site of a Cys-free mutant of the enzyme, which were subsequently alkylated with 1-iodoalkanes. By different combinations of site-specific mutations and chemical modifications of the enzyme the enantioselectivity in the conjugation of glutathione with the epoxide-containing substrates 1-phenylpropylene oxide and styrene-7,8-oxide were enhanced up to 9- and 10-fold. The results also demonstrate that the enantioselectivity can be diminished, or even reversed, by suitable modifications, which can be valuable under some conditions. The redesign of the active-site structure for enhanced or diminished enantioselectivities have divergent requirements for different epoxides, calling for a combinatorial approach involving alternative mutations and chemical modifications to optimize the enantioselectivity for a targeted substrate. This approach outlines a general method of great potential for fine-tuning substrate specificity and tailoring stereoselectivity of recombinant enzymes.

Place, publisher, year, edition, pages
2007. Vol. 1770, no 9, 1374-1381 p.
Keyword [en]
Enantioselectivity, Epoxide resolution, Glutathione transferase, Protein modification, Rational redesign
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-11976DOI: 10.1016/j.bbagen.2007.06.002ISI: 000249511200014PubMedID: 17689871OAI: oai:DiVA.org:uu-11976DiVA: diva2:39745
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Modulating Enzyme Functions by Semi-Rational Redesign and Chemical Modifications: A Study on Mu-class Glutathione Transferases
Open this publication in new window or tab >>Modulating Enzyme Functions by Semi-Rational Redesign and Chemical Modifications: A Study on Mu-class Glutathione Transferases
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Today, enzymes are extensively used for many industrial applications, this includes bulk and fine-chemical synthesis, pharmaceuticals and consumer products. Though Nature has perfected enzymes for many millions of years, they seldom reach industrial performance targets. Natural enzymes could benefit from protein redesign experiments to gain novel functions or optimize existing functions.

Glutathione transferases (GSTs) are detoxification enzymes, they also display disparate functions. Two Mu-class GSTs, M1-1 and M2-2, are closely related but display dissimilar substrate selectivity profiles. Saturation mutagenesis of a previously recognized hypervariable amino acid in GST M2-2, generated twenty enzyme variants with altered substrate selectivity profiles, as well as modified thermostabilities and expressivities. This indicates an evolutionary significance; GST Mu-class enzymes could easily alter functions in a duplicate gene by a single-point mutation.

To further identify residues responsible for substrate selectivity in the GST M2-2 active site, three residues were chosen for iterative saturation mutagenesis. Mutations in position10, identified as highly conserved, rendered enzyme variants with substrate selectivity profiles resembling that of specialist enzymes. Ile10 could be conserved to sustain the broad substrate acceptance displayed by GST Mu-class enzymes.

Enzymes are constructed from primarily twenty amino acids, it is a reasonable assumption that expansion of the amino acid repertoire could result in functional properties that cannot be accomplished with the natural set of building blocks. A combination approach of site-directed mutagenesis and chemical modifications in GST M2-2 and GST M1-1 resulted in novel enzyme variants that displayed altered substrate selectivity patterns as well as improved enantioselectivities.

The results presented in this thesis demonstrate the use of different protein redesign techniques to modulate various functions in Mu-class GSTs. These techniques could be useful in search of optimized enzyme variants for industrial targets.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 741
Keyword
protein redesign, semi-rational redesign, saturation mutagenesis, iterative saturation mutagenesis, chemical modification, Cys, Cys-X scanning, enzyme evolution, promiscuous, substrate selectivity, enantioselectivity
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-149326 (URN)978-91-554-8029-5 (ISBN)
Public defence
2011-04-29, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Note
biokemi och organisk kemiAvailable from: 2011-04-08 Created: 2011-03-17 Last updated: 2011-05-05Bibliographically approved

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Ivarsson, YlvaNorrgård, Malena A.Hellman, UlfMannervik, Bengt

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