Genetic Selection of Novel Chorismate Mutases

It is in the realm of very large combinatorial libraries that selection rather than screening gains crucial importance. As the focus shifts from randomizing an eight-residue peptide to a 100 amino acid protein (the typical size of a small functional domain, for example a chorismate mutase domain), the number of sequence permutations rises to an astronomical 20100. The ability to assay even a tiny fraction of this sequence space in directed molecular evolution experiments demands selection, even though initial development of an appropriate system may be considerably more involved than the setup of a screening procedure. 

The utilization of evolutionary strategies in the laboratory can be illustrated with proteins that catalyze simple metabolic reactions. One of the simplest such reactions is the conversion of chorismate to prephenate (Fig. 3.3), a [3,3]-sigmatropic rearrangement. This transformation is a key step in the shikimate pathway leading to aromatic amino acids in plants and lower organisms [28, 29]. It is accelerated more than a million-fold by enzymes called chorismate mutases [30], 

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