Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications.
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.
Ivarsson
Ylva
Y
Department of Biochemistry and Organic Chemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden.
Norrgård
Malena A
MA
Hellman
Ulf
U
Mannervik
Bengt
B
D000475:Alkenes / Q000737:chemistry; D000478:Alkylation; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D004852:Epoxy Compounds / Q000737:chemistry; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013237:Stereoisomerism; D013267:Stilbenes / Q000737:chemistry
D000475:Alkenes; D004852:Epoxy Compounds; D013267:Stilbenes; C509597:phenylpropylene oxide; C013690:styrene oxide; D005982:Glutathione Transferase; D003545:Cysteine; C025906:stilbene oxide