Redesign of substrate specificity of glutathione transferase and glutathione reductase : Enzyme engineering by directed mutagenesis, phage-display selection and DNA shuffling

Sammanfattning: Human glutathione transferase (GST) A1-1 was expressed in fusion with the phage gene IIIproduct and a library of phage-displayed GST mutants was constructed by randomization of fouractive-site residues. Variant GSTs were isolated by mechanism-based phage display selection, using an immobilized transition-state analog of a nucleophilic aromatic substitution (SNAr) reaction. One isolated mutant exhibited a 105-fold rate enhancement with the SNAr substrate l-chloro-2,4-dinitrobenzene (CDNB) compared to the uncatalyzed reaction. This mutant was further randomized, in a fifth active-site residue to further improve the catalytic efficiency. Activity screening of 18 GST variants identified one mutant, Met208Cys, that exhibited a 2-fold enhanced catalytic efficiency with CDNB.A library of chimeric random mutants was constructed by shuffling of cDNA encoding thehomologous human enzymes GST M1-1 and GST M2-2. Activity screening and DNA sequence analysis of sampled mutants provided information about the differential substrate specificities withaminochrome and 2-cyano-1,3-dimethyl-1-nitrosoguanidine of the two wild-type isoenzymes.Chimeric enzyme variants derived from the DNA-shuffled library were further analyzed together with point mutants having residues in GST M1-1 replaced by those of GST M2-2. The results indicated that Arg165 in human GST M2-2 is important for the substitution reactions with 2-cyano-1,3-dimethyl-l-nitrosoguanidine and 1,2-dichloro-4-nitrobenzene.The three-dimensional structure of Anabaena PCC 7120 glutathione reductase was modeled based on a sequence alignment with the Escherichia coli and human enzymes, for which crystalstructures are known. The model was used for redesigning the enzyme for structure-activity studies. Removal of a 10-residue loop, predicted to be situated next to the entrance of the pyridine-nucleotide-binding site, increased the catalytic efficiency with both coenzymes NADPH and NADH.