Remodeling enzyme active sites by stepwise loop insertion

The remolding active site loops via residue insertion/deletion as well as substitution is thought to play a key role in enzyme divergent evolution. However, enzyme engineering by residue insertion in active site loops often severely perturbs the protein structural integrity and causes protein misfolding and activity loss. We have designed a stepwise loop insertion strategy (StLois), in which a pair of randomized residues is introduced in a stepwise manner, efficiently collating mutational fitness effects. The strategy of StLois constitutes three key steps. First, the target regions should be identified through structural and functional analysis on the counterpart enzymes. Second, pair residues can be introduced in loop regions through insertion with NNK codon degeneracy. Third, the best hit used as a template for the next round mutagenesis. The residue insertion process can repeat as many times as necessary. By using the StLois method, we have evolved the substrate preference of a lactonase to phosphotriesterase. In this chapter, we describe the detailed StLois technique, which efficiently expands the residue in the loop region and remolds the architecture of enzyme active site for novel catalytic properties.