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- Ivarsson, Ylva, et al.
(författare)
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Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications
- 2007
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Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 0304-4165 .- 1872-8006. ; 1770:9, s. 1374-1381
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Ivarsson, Ylva, 1976-
(författare)
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Evolutionary Analysis and Posttranslational Chemical Modifications in Protein Redesign : A Study on Mu Class Glutathione Transferases
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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- Ivarsson, Ylva, et al.
(författare)
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Regio- and enantioselectivities in epoxide conjugations are modulated by residue 210 in Mu class glutathione transferases.
- 2005
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Ingår i: Protein Eng Des Sel. - 1741-0126. ; 18:12, s. 607-16
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Tidskriftsartikel (refereegranskat)abstract
- The homologous human glutathione transferases (GSTs) M1-1 and M2-2 have similar catalytic activities with many electrophilic substrates, but differ strikingly in their conjugation of epoxides with glutathione. Residue 210, Thr in GST M2-2 and Ser in GST M1-1, is a key active-site component in determining the activity profile with epoxide substrates. This residue is hypervariable in Mu class GSTs, suggesting that it has special significance in the evolution of new functions. The present study shows that minor modifications of this residue can have major consequences for the enzyme-catalyzed epoxide conjugations. In general, a Ser at position 210 gives the highest catalytic efficiency, but the relatively high activity with an Ala placed on this position demonstrates that a hydroxyl group is not required. In contrast, a Thr residue suppresses the activity with epoxides by several orders of magnitude without major effects on the activity with alternative GST substrates. Residue 210 influences both the regio- and enantioselectivity with chiral and prochiral epoxides of stilbene and styrene and influences the distribution of isomeric glutathione conjugates. Thus, residue 210 contributes to both stereoselective recognition of the substrates and to partitioning of the isomeric reactants to the alternative transition states leading to separate chiral products.
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