title: Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold.
abstract: 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.
authors: 152 Davey Laboratory, Department of Chemistry, Pennsylvania State University, University Park, PA 16802-6300, USA.
authors: A E
authors: AE
authors: Nixon
authors: S M
authors: SM
authors: Firestine
authors: F G
authors: FG
authors: Salinas
authors: S J
authors: SJ
authors: Benkovic
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D015195:Drug Design; D006836:Hydro-Lyases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C082741:scytalone dehydratase