TY - JOUR
T1 - Rational design of a scytalone dehydratase-like enzyme using a structurally homologous protein scaffold
AU - Nixon, Andrew E.
AU - Firestine, Steven M.
AU - Salinas, Frank G.
AU - Benkovic, Stephen J.
PY - 1999/3/30
Y1 - 1999/3/30
N2 - 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 μM, 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.
AB - 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 μM, 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.
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U2 - 10.1073/pnas.96.7.3568
DO - 10.1073/pnas.96.7.3568
M3 - Article
C2 - 10097077
AN - SCOPUS:0033616574
SN - 0027-8424
VL - 96
SP - 3568
EP - 3571
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 7
ER -