Evolution of differential substrate specificities in Mu class glutathione transferases probed by DNA shuffling

A library of variant enzymes was created by combined shuffling of the DNA e ncoding the human Mu class glutathione transferases GST M1-1 and GST M2-2. The parental GSTs are 84% sequence identical at the protein level, but thei r specific activities with the substrates aminochrome and 2-cyano-1,3-dimet hyl-1-nitrosoguanidine (cyanoDMNG) differ by more than 100-fold. Aminochrom e is of particular interest as an oxidation product of dopamine and of poss ible significance in the etiology of Parkinson's disease, and cyanoDMNG is a model for genotoxic and potentially carcinogenic nitroso compounds. GST M 2-2 has at least two orders of magnitude higher catalytic activity with bot h of the substrates than any of the other known GSTs, including GST M1-1. T he DNA library of variant Mu class GST sequences contained "mosaic" structu res composed of alternating segments of both parental sequences. All clones contained the 5'-end of a GST M1-1 clone optimized for high-level expressi on in Escherichia coli. The remainder of the sequences derived from segment s of GST M2-2 and GST M1-1 DNA. All of the clones analyzed contained betwee n two and seven distinct DNA segments. In addition, each clone contained an average of approximately one point mutation. None of the library clones an alyzed was identical with either of the two parental structures. Variant GS T sequences were expressed in E. coil, and their enzymatic activities with aminochrome, cyanoDMNG, and 1-chloro-2,4-dinitrobenzene (CDNB) were determi ned in bacterial lysates. Such screening of more than 70 clones demonstrate d a continuous range of activities covering at least two orders of magnitud e for each of the substrates. For a given clone, the activities with aminoc hrome and cyanoDMNG, in spite of their different chemistries, were clearly correlated, whereas no strong correlation was found with CDNB. This functio nal correlation suggests a common structural basis for Be enzymatic mechani sms for conjugation of aminochrome and denitrosation of cyanoDMNG. From an evolutionary perspective, the results show that recombination of segments f rom homologous proteins gives rise to a large proportion of functionally co mpetent proteins with a range of activities. The data support the proposal that natural evolution of protein functions may involve recombination of: D NA segments followed by selection for advantageous functional properties of the resulting proteins. Clearly, the same approach can be utilized in the engineering of proteins displaying novel functions by in vitro evolution. ( C) 1999 Academic Press.