Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold

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