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 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.
Nixon
A E
AE
152 Davey Laboratory, Department of Chemistry, Pennsylvania State University, University Park, PA 16802-6300, USA.
Firestine
S M
SM
Salinas
F G
FG
Benkovic
S J
SJ
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
D002352:Carrier Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C082741:scytalone dehydratase