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Cys-X Scanning for Expansion of Active-site Residues and Modulation of Catalytic Functions in a Glutathione Transferase
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.
2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 19, 16871-16878 p.Article in journal (Refereed) Published
Abstract [en]

We propose Cys-X scanning as a semisynthetic approach to engineer the functional properties of recombinant proteins. As in the case of Ala scanning, key residues in the primary structure are identified, and one of them is replaced by Cys via site-directed mutagenesis. The thiol of the residue introduced is subsequently modified by alternative chemical reagents to yield diverse Cys-X mutants of the protein. This chemical approach is orthogonal to Ala or Cys scanning and allows the expansion of the repertoire of amino acid side chains far beyond those present in natural proteins. In its present application, we have introduced Cys-X residues in human glutathione transferase (GST) M2-2, replacing Met-212 in the substrate-binding site. To achieve selectivity of the modifications, the Cys residues in the wild-type enzyme were replaced by Ala. A suite of simple substitutions resulted in a set of homologous Met derivatives ranging from normethionine to S-heptyl-cysteine. The chemical modifications were validated by HPLC and mass spectrometry. The derivatized mutant enzymes were assayed with alternative GST substrates representing diverse chemical reactions: aromatic substitution, epoxide opening, transnitrosylation, and addition to an ortho-quinone. The Cys substitutions had different effects on the alternative substrates and differentially enhanced or suppressed catalytic activities depending on both the Cys-X substitution and the substrate assayed. As a consequence, the enzyme specificity profile could be changed among the alternative substrates. The procedure lends itself to large-scale production of Cys-X modified protein variants.

Place, publisher, year, edition, pages
The American Society for Biochemistry and Molecular Biology, Inc. , 2011. Vol. 286, no 19, 16871-16878 p.
Keyword [en]
protein redesign, chemical modification, substrate selectivity, Cys, enzyme evolution, glutathione transferases, GST M2-2, Cys-X scanning
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-149319DOI: 10.1074/jbc.M111.230078ISI: 000290301900036PubMedID: 21454564OAI: oai:DiVA.org:uu-149319DiVA: diva2:404543
Available from: 2011-03-17 Created: 2011-03-17 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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