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Programmed Delivery of Novel Functional Groups to the Alpha Class Glutathione Transferases
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.
2003 (English)In: Biochemistry, ISSN 0006-2960, Vol. 42, no 34, 10260-10268 p.Article in journal (Refereed) Published
Abstract [en]

Here we describe a new route to site- and class-specific protein modification that will allow us to create novel functional proteins with artificial chemical groups. Glutathione transferases from the alpha but not the mu, pi, omega, or theta classes can be rapidly and site-specifically acylated with thioesters of glutathione (GS-thioesters) that are similar to compounds that have been demonstrated to occur in vivo. The human isoforms A1-1, A2-2, A3-3, and A4-4 from the alpha class all react with the reagent at a conserved tyrosine residue (Y9) that is crucial in catalysis of detoxication reactions. The yield of modified protein is virtually quantitative in less than 30 min under optimized conditions. The acylated product is stable for more than 24 h at pH 7 and 25 °C. The modification is reversible in the presence of excess glutathione, but the labeled protein can be protected by adding S-methylglutathione. The stability of the ester with respect to added glutathione depends on the acyl moiety. The reaction can also take place in Escherichia coli lysates doped with alpha class glutathione transferases. A control substance that lacks the peptidyl backbone required for binding to the glutathione transferases acylates surface-exposed lysines. There is some acyl group specificity since one out of the three different GS-thioesters that we tried was not able to acylate Y9.

Place, publisher, year, edition, pages
2003. Vol. 42, no 34, 10260-10268 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-13363DOI: 10.1021/bi0343525OAI: oai:DiVA.org:liu-13363DiVA: diva2:20486
Available from: 2005-09-23 Created: 2005-09-23 Last updated: 2017-10-27
In thesis
1. A Novel Route for Construction of Multipurpose Receptors through Chemical Modification of Glutathione Transferases
Open this publication in new window or tab >>A Novel Route for Construction of Multipurpose Receptors through Chemical Modification of Glutathione Transferases
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes how the human Alpha class glutathione transferase (GST) A1-1 can be reprogrammed either to function as a multipurpose biosensor for detection of small molecule analytes, or as a handle providing for more efficient protein purification.

A novel, user-friendly, and efficient method for site-specific introduction of functional groups into the active site of hGST A1-1 is the platform for these achievements. The designed thioester reagents are glutathione-based and they are able to label one single nucleophile (Y9) and leave the other 50 nucleophiles (in hGST A1-1) intact. The modification reaction was tested with five classes of GSTs (Alpha, Mu, Pi, Theta and Omega) and was found to be specific for the Alpha class isoenzymes. The reaction was further refined to target a single lysine residue, K216 in the hGST A1-1 mutant A216K, providing a stable amide bond between the protein and the labeling group. To further improve the labeling process, biotinylated reagents that could deliver the acyl group to Y9 (wt hGST A1-1) or K216 in the lysine mutant, while attached to streptavidin-coated agarose beads, were designed and synthesized.

A focused library of eleven A216K/M208X mutants was made via random mutagenesis to provide an array of proteins with altered micro-environments in the hydrophobic binding site, where M208 is situated. Through the invented route for site-specific labeling, a fluorescent probe (coumarin) was introduced on K216 in all double mutants, with the purpose of developing a protein-based biosensor, akin to the olfactory system. The array of coumarin-labeled proteins responded differently to the addition of different analytes, and the responses were analyzed through pattern recognition of the fluorescence signals. The labeled proteins could also be site-specifically immobilized on a PEG-based biosensor chip via the single C112 on the surface of the protein, enabling development of surface-based biosensing systems.

Also, a refined system for efficient detection and purification of GST-fusion proteins is presented. Through a screening process involving A216K and all produced A216K/M208X mutants, two candidates (A216K and A216K/M208F) were singled out as scaffolds for the next generation of fusion proteins. In addition to the features present in commercially available GST fusion constructs, the new mutants can be site-specifically labeled with a fluorophore in bacterial lysates providing quick and sensitive monitoring of expression and purification. Furthermore, the proteins could be labeled with a unique aldehyde moiety providing for a novel protein purification scheme.

Place, publisher, year, edition, pages
Institutionen för teknik och naturvetenskap, 2008. 79 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1184
Keyword
Human GST A1-1, site-specific covalent modification, tyrosine 9, lysine 216, methionine 208, multipurpose receptor, pattern recognition, protein purification
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-11612 (URN)978-91-7393-893-8 (ISBN)
Public defence
2008-05-23, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2008-04-28 Created: 2008-04-28 Last updated: 2009-05-18
2. Catalysis and Site-Specific Modification of Glutathione Transferases Enabled by Rational Design
Open this publication in new window or tab >>Catalysis and Site-Specific Modification of Glutathione Transferases Enabled by Rational Design
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the rational design of a novel enzyme, a thiolester hydrolase, derived from human glutathione transferase (GST) A1-1 by the introduction of a single histidine residue. The first section of the thesis describes the design and the determination of the reaction mechanism. The design was based on the crystal structure of human GST A1-1 complexed with S-benzylglutathione. The resulting enzyme, A216H, catalyzed the hydrolysis of the non-natural substrate GSB, a thiolester of glutathione and benzoic acid. The reaction followed saturation kinetics with a kcat of 0.00078 min-1 and KM of 5 μM. The rate constant ratio, (kcat/KM)/kuncat, was found to be more than 107 M-1. The introduction of a single His residue in position 216 opened up a novel reaction pathway in human GST A1-1 and is a nice example of catalytic promiscuity. The substrate requirements were investigated and A216H was found to be selective since only two out of 18 GS-thiolesters tested were substrates for A216H. The reaction mechanism of the A216H-catalyzed hydrolysis of GSB was determined and found to proceed via an acyl intermediate at Y9. The hydrolysis was catalyzed by H216 that acts as a general base and the deacylation was found to be the rate-determining step. The Y9-intermediate could be selectively trapped by oxygen nucleophiles and primary alcohols, in particular 1-propanol and trifluoroethanol, were the most efficient. In addition, saturation kinetics was obtained in the acyl transfer reaction with 1-propanol indicating the presence of a second binding site in A216H.

The second section of this thesis describes the site-specific covalent modification of human GST A1-1. The addition of GSB to the wild-type protein results in a site-specific benzoylation of only one tyrosine residue, Y9, out of ten present in the protein (one out of totally 51 nucleophiles). The reaction was tested with five GST classes (Alpha, Mu, Pi, Theta and Omega) and found to be specific for the Alpha class isoenzymes. The covalent modification reaction was further refined to target a single lysine residue, K216, providing a more stable linkage in the form of an amide bond. The reaction was found to be versatile and approximately 50% of the GS-thiolesters tested acylated K216, including a fluorophore.

Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi, 2005
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 964
Keyword
Catalysis, Glutathione Transferases, Rational design, Protein design, Site-specific modification, thiolester hydrolysis
National Category
Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:liu:diva-3962 (URN)91-85457-09-4 (ISBN)
Public defence
2005-09-30, 09:15 (English)
Supervisors
Note

On the day of the public defence the status of article II was: Submitted and article IV was: In press.

Available from: 2005-09-23 Created: 2005-09-23 Last updated: 2017-10-27

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Håkansson Hederos, SofiaViljanen, JohanBroo, Kerstin

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