Redesign of substrate specificity of glutathione transferase and glutathione reductase: Enzyme engineering by directed mutagenesis, phage-display selection and DNA shuffling
1999 (English)Doctoral thesis, comprehensive summary (Other academic)
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.
Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 1999. , 48 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 450
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:uu:diva-971ISBN: 91-554-4458-XOAI: oai:DiVA.org:uu-971DiVA: diva2:173309
1999-05-08, lecture hall B42, Biomedical Center, Uppsala, Uppsala, 10:15