Directed Enzyme Evolution of Theta Class Glutathione Transferase : Studies of Recombinant Libraries and Enhancement of Activity toward the Anticancer Drug 1,3-bis(2-Chloroethyl)-1-nitrosourea

Sammanfattning: Glutathione transferases (GSTs) are detoxication enzymes involved in the cellular protection against a wide range of reactive substances. The role of GSTs is to catalyze the conjugation of glutathione with electrophilic compounds, which generally results in less toxic products. The ability to catalyze the denitrosation of the anticancer drug 1,3-bis(2-chloroethyl)- 1-nitrosourea (BCNU) was measured in twelve different GSTs. Only three of the enzymes showed any measurable activity with BCNU, of which human GST T1-1 was the most efficient. This is of special interest, since human GST T1-1 is a polymorphic protein and its expression in different patients may be crucial for the response to BCNU.DNA shuffling was used to create a mutant library by recombination of cDNA coding for two different Theta-class GSTs. In total, 94 randomly picked mutants were characterized with respect to their catalytic activity with six different substrates, expression level and sequence. A clone with only one point mutation compared to wild-type rat GST T2-2 had a significantly different substrate-activity pattern. A high expressing mutant of human GST T1-1 was also identified, which is important, since the yield of the wild-type GST T1-1 is generally low. Characterization of the Theta library demonstrated divergence of GST variants both in structure and function. The properties of every mutant were treated as a point in a six-dimensional substrate-activity space. Groups of mutants were formed based on euclidian distances and K-means cluster analyses. Both methods resulted in a set of five mutants with high alkyltransferase activities toward dichloromethane and 4-nitrophenethyl bromide (NPB). The five selected mutants were used as parental genes in a new DNA shuffling. Addition of cDNA coding for mouse and rat GST T1-1 improved the genetic diversity of the library. The evolution of GST variants was directed towards increased alkyltransferase activity including activity with the anticancer drug BCNU. NPB was used as a surrogate substrate in order to facilitate the screening process. A mutant from the second generation displayed a 65-fold increased catalytic activity with NPB as substrate compared to wild-type human GST T1-1. The BCNU activity with the same mutant had increased 175-fold, suggesting that NPB is a suitable model substrate for the anticancer drug. Further evolution presented a mutant in the fifth generation of the library with 110 times higher NPB activity than wild-type human GST T1-1.