Reetz_2005_Angew.Chem.Int.Ed.Engl_44_4192

Herein, we describe a method for expanding the scope of substrate acceptance of a given enzyme with the aim of including a wide range of structurally different compounds. Two straightforward steps are required: the design and the generation of relatively small focused libraries of enzyme mutants produced by randomization at several sets of two spatially close amino acid positions around the active site. The choice of two amino acids which are spatially close to one another allows for potential synergistic conformational effects arising from side-chain orientations, an unpredictable phenomenon which cannot be brought about by single-site saturation mutagenesis. The optimal choice of the respective pairs of amino acids is guided by the 3D structure of the wild-type (WT) enzyme with a bound substrate. Geometric inspection allows the definition of sites at which the side chains of the individual amino acids in each pair point toward the binding site of the WT enzyme