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Updated: Jul 13, 2026

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Published on: October 4, 2017
Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical
Ylva Ivarsson1, Malena A Norrgård, Ulf Hellman
1Department of Biochemistry and Organic Chemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden.
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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