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 k(cat) 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 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.
机构:
Univ Calif San Francisco, Sch Pharm, Dept Pharmaceut Chem, San Francisco, CA 94143 USAUniv Calif San Francisco, Sch Pharm, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
Babbitt, PC
Gerlt, JA
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机构:Univ Calif San Francisco, Sch Pharm, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
机构:
Univ Calif San Francisco, Sch Pharm, Dept Pharmaceut Chem, San Francisco, CA 94143 USAUniv Calif San Francisco, Sch Pharm, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
Babbitt, PC
Gerlt, JA
论文数: 0引用数: 0
h-index: 0
机构:Univ Calif San Francisco, Sch Pharm, Dept Pharmaceut Chem, San Francisco, CA 94143 USA