Design of the active site architecture of an enzyme for evolution of new catalytic function
Hak-Sung Kim

The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we present strategies to design active site architecture of enzyme for evolution of new catalytic function. To this end, we developed a new process, termed simultaneous incorporation and adjustment of functional elements (SIAFE), comprising the insertion, deletion, and substitution of functional elements followed by point mutations for the redesign of an enzyme’s active site architecture to evolve another activity. By applying this approach to the ??/?? metallo hydrolase scaffold of glyoxalase II, we were able to introduce ?-lactamase activity into this scaffold. The resulting enzyme, evMBL8, was found to have completely lost its original activity and, instead, catalyzes the hydrolysis of cefotaxime, thus conferring an approximately 100-fold increase in resistance to Escherichia coli growth on cefotaxime. The site-directed mutagenesis and molecular modeling of evMBL8 support a rationale for metal coordination and substrate binding at the active site. As the second approach, a modulation of the substrate specificity of a ?/? barrel enzyme was conducted by structure-based design of the active site loops. The active site of the enzyme was docked with a target substrate, and the critical residues on the active site-constituting three loops were selected. Mutant enzyme showing highest specificity toward the target substrate was successfully generated through site-directed and saturation mutagenesis. These approaches provide general strategies that may be applicable to the creation of enzymes with new functions from existing scaffolds.