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Identification of residues in glutathione transferase capable of driving functional diversification in evolution: A novel approach to protein redesign
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
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2003 In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 278, no 10, 8733-8738 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2003. Vol. 278, no 10, 8733-8738 p.
URN: urn:nbn:se:uu:diva-94455OAI: oai:DiVA.org:uu-94455DiVA: diva2:168302
Available from: 2006-04-28 Created: 2006-04-28 Last updated: 2013-07-09Bibliographically approved
In thesis
1. Evolutionary Analysis and Posttranslational Chemical Modifications in Protein Redesign: A Study on Mu Class Glutathione Transferases
Open this publication in new window or tab >>Evolutionary Analysis and Posttranslational Chemical Modifications in Protein Redesign: A Study on Mu Class Glutathione Transferases
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates.

Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity.

To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations.

The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide.

The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 54 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 178
Biochemistry, glutathione transferase, evolutionary analysis, positive selection, epoxide, stereoselectivity, protein modification, Biokemi
urn:nbn:se:uu:diva-6840 (URN)91-554-6557-9 (ISBN)
Public defence
2006-05-19, B22, BMC, Uppsala, 10:15
Available from: 2006-04-28 Created: 2006-04-28Bibliographically approved

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