A novel quasi-species of glutathione transferase with high activity towards naturally occurring isothiocyanates evolves from promiscuous low-activity variants
2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 401, no 3, 451-464 p.Article in journal (Refereed) Published
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 the high activity for substrates distinguishing the 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 the recombinant enzyme variants were active with at least one of three alternative substrates: phenethyl isothiocyanate (PEITC), 1-chloro-2,4-dinitrobenzene (CDNB), or p-nitrophenyl acetate (pNPA). 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 them 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 comprising variant amino acid sequences. The quasi-species are structurally distinguished by the active-site residues that govern their substrate selectivities. Clearly, minimal alterations of the active site can generate enzymes with highly distinctive functional properties.
Place, publisher, year, edition, pages
2010. Vol. 401, no 3, 451-464 p.
functional quasi-species, glutathione transferase, substrate matrix, multivariate analysis, isothiocyanates, activity profiles
IdentifiersURN: urn:nbn:se:uu:diva-122377DOI: 10.1016/j.jmb.2010.06.033ISI: 000281171000010PubMedID: 20600121OAI: oai:DiVA.org:uu-122377DiVA: diva2:309931
Uppdaterad från Manuskript till Artikel 201012032010-04-092010-04-092010-12-03Bibliographically approved