A Novel Quasi-Species of Glutathione Transferase with High Activity towards Naturally Occurring Isothiocyanates Evolves from Promiscuous Low-Activity Variants |
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Authors: | Arna Runarsdottir |
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Affiliation: | Department of Biochemistry and Organic Chemistry, Uppsala University, Biomedical Center, Box 576, SE-75123 Uppsala, Sweden |
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Abstract: | Glutathione transferases (GSTs) are known as promiscuous enzymes capable of catalyzing the conjugation of glutathione with a broad range of electrophilic substrates. A previous study based on recombinant chimeras derived from human GST M1-1 and GST M2-2 demonstrated the formation of a subset of F1 generation GSTs, which had lost high activity with substrates distinguishing parental enzymes. In the present study, the members of this subset were recombined by DNA shuffling to produce an F2 generation of GSTs. Screening of 930 bacterial clones demonstrated that 83% of recombinant enzyme variants were active with at least one of three alternative substrates: phenethyl isothiocyanate (PEITC), 1-chloro-2,4-dinitrobenzene, or p-nitrophenyl acetate. The majority had similar low activity as the parental GSTs in the F1 generation. However, 17 novel enzymes displayed high activity with PEITC. Half of these enzymes were similar to GST M1-1, which also has high activity with the same substrate, and all of these GSTs featured Tyr116/Ser210 in the active site. This group of F2 variants apparently had reverted to the GST M1-1 type. A second group of F2 variants with high PEITC activity was characterized by His116 in the active site. This category represented a new variety of GSTs, which demonstrated higher selectivity for isothiocyanate substrates than the GST M1-1 type. The different groups of GSTs can be considered as distinct molecular quasi-species, each of which comprises variant amino acid sequences. The quasi-species are structurally distinguished by active-site residues that govern their substrate selectivities. Clearly, minimal alterations of the active site can generate enzymes with highly distinctive functional properties. |
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Keywords: | GST, glutathione transferase PEITC, phenethyl isothiocyanate CDNB, 1-chloro-2,4-dinitrobenzene pNPA, p-nitrophenyl acetate ITC, isothiocyanate EPNP, epoxy-3-(4-nitrophenoxy)-propane PCA, principal component analysis NPG, 3-(4-nitrophenyl)-glycidol benzyl-ITC, benzyl isothiocyanate propyl-ITC, propyl isothiocyanate cyclohexyl-ITC, cyclohexyl isothiocyanate allyl-ITC, allyl isothiocyanate tPBO, trans-4-phenyl-3-buten-2-one |
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