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

被引:25
作者
Nixon, AE [1 ]
Firestine, SM [1 ]
Salinas, FG [1 ]
Benkovic, SJ [1 ]
机构
[1] Penn State Univ, Dept Chem, University Pk, PA 16802 USA
关键词
D O I
10.1073/pnas.96.7.3568
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
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页码:3568 / 3571
页数:4
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