RESUMO
The generation of enzymes to catalyze specific reactions is one of the more challenging problems facing protein engineers. Structural similarities between the enzyme scytalone dehydratase with nuclear transport factor 2 (NTF2) suggested the potential for NTF2 to be re-engineered into a scytalone dehydratase-like enzyme. We introduced four key catalytic residues into NTF2 to create a scytalone dehydratase-like active site. A C-terminal helix found in scytalone dehydratase but absent in NTF2 also was added. Mutant NTF2 proteins were tested for catalytic activity by using a spectroscopic assay. One of the engineered enzymes exhibited catalytic activity with minimal kcat and Km values of 0.125 min-1 and 800 microM, respectively. This level of catalytic activity represents minimally a 150-fold improvement in activity over the background rate for substrate dehydration and a dramatic step forward from the catalytically inert parent NTF2. This work represents one of the few examples of converting a protein scaffold into an enzyme, outside those arising from the induction of catalytic activity into antibodies.
