Glutathione transferases (GSTs) provide an excellent platform for investigations of redesign of proteins for novel functions. In Nature higher eukaryotes display numerous active genes. Humans have up to 17 different genes, Drosophila melanogaster 37, and poplars have 81. Sequence homologies indicate that the genes have divergently evolved from a common ancestor. The significance of the multiple forms is that they shared different substrate selectivities, even though some of them display overlapping activities. Evidently, the redesign of a given enzyme can give rise to multiple variants with different activities. Our laboratory has demonstrated that mutations of a small number of amino acid residues by protein engineering can give rise to GSTs that have gained novel catalytic properties. The stochastic approach of generating mutant libraries by shuffling of DNA encoding different GSTs has also given rise to large numbers of catalytically competent GSTs. In some instances the mutants form clusters of functionally related variants similar to isoenzymes of an extant GST. In other cases clusters display functional properties that distinguish them significantly from the parental forms. We have adopted the notion of quasi-species to designate such clusters and we propose that a cluster should be subjected to directed evolution rather than the single most active variant.
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Molecular Quasi-Species in Evolution Applied to the Engineering of Promiscuous Glutathione Transferases
Bengt Mannervik
