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

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
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 13, 4876-4881 p.Article in journal (Refereed) Published
Abstract [en]

All molecular species in an organism are connected physically and functionally to other molecules. In evolving systems, it is not obvious to what extent functional properties of a protein can change to selective advantage and leave intact favorable traits previously acquired. This uncertainty has particular significance in the evolution of novel pathways for detoxication, because an organism challenged with new xenobiotics in the environment may still require biotransformation of previously encountered toxins. Positive selection has been proposed as an evolutionary mechanism for facile adaptive responses of proteins to changing conditions. Here, we show, by saturation mutagenesis, that mutations of a hypervariable residue in human glutathione transferase M2-2 can differentially change the enzyme's substrate-activity profile with alternative substrates and, furthermore, enable or disable dissimilar chemical reactions. Crystal structures demonstrate that activity with epoxides is enabled through removal of steric hindrance from a methyl group, whereas activities with an orthoquinone and a nitroso donor are maintained in the variant enzymes. Given the diversity of cellular activities in which a single protein can be engaged, the selective transmutation of functional properties has general significance in molecular evolution.

Place, publisher, year, edition, pages
2006. Vol. 103, no 13, 4876-4881 p.
Keyword [en]
Enzyme Stability, Epoxy Compounds/metabolism, Evolution; Molecular, Glutathione Transferase/chemistry/*genetics/*metabolism, Humans, Models; Molecular, Molecular Sequence Data, Molecular Structure, Mutation/genetics, Protein Structure; Tertiary, Research Support; Non-U.S. Gov't, Serine/chemistry/metabolism, Substrate Specificity, Temperature, Threonine/chemistry/metabolism, Variation (Genetics)/genetics
Identifiers
URN: urn:nbn:se:uu:diva-81516DOI: 10.1073/pnas.0600849103PubMedID: 16549767OAI: oai:DiVA.org:uu-81516DiVA: diva2:109431
Available from: 2008-01-29 Created: 2008-01-29 Last updated: 2017-12-14Bibliographically 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

Open Access in DiVA

No full text

Other links

Publisher's full textPubMedhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Retrieve&list_uids=16549767&dopt=Citation

Authority records BETA

Ivarsson, YlvaMannervik, Bengt

Search in DiVA

By author/editor
Ivarsson, YlvaMannervik, Bengt
By organisation
Department of Biochemistry and Organic ChemistryStructural Molecular Biology
In the same journal
Proceedings of the National Academy of Sciences of the United States of America

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 925 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf