Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications

• Published in: Biochimica et biophysica acta Vol. 1770; no. 9; pp. 1374 - 1381
• Main Authors: Ivarsson, Ylva, Norrgård, Malena A., Hellman, Ulf, Mannervik, Bengt
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2007
• Subjects: Alkenes - chemistry; Alkylation; Amino Acid Sequence; Binding Sites - genetics; Chemistry; Cysteine - chemistry; Enantioselectivity; Epoxide resolution; Epoxy Compounds - chemistry; Glutathione transferase; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Humans; Kemi; Kinetics; Mutagenesis, Site-Directed - methods; NATURAL SCIENCES; NATURVETENSKAP; Protein Engineering - methods; Protein modification; Rational redesign; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stereoisomerism; Stilbenes - chemistry; Enantioselectivity; Glutathione transferase; Epoxide resolution; GST; tSBO; CDNB; SO; GSH; PPO; Rational redesign; Protein modification
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2007.06.002

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.