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Science 27 January 2006:
Vol. 311. no. 5760, pp. 535 - 538
DOI: 10.1126/science.1118953
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Reports

Design and Evolution of New Catalytic Activity with an Existing Protein Scaffold

Hee-Sung Park,1 Sung-Hun Nam,1 Jin Kak Lee,2* Chang No Yoon,2 Bengt Mannervik,3 Stephen J. Benkovic,4 Hak-Sung Kim1{dagger}

The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce ß-lactamase activity into the {alpha}ß/ß{alpha} metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo ß-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (kcat /Km)app of 1.8 x 102 (mole/liter)–1 second–1, thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.

1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-Dong, Yusung-Gu, Daejon 305-701, Korea.
2 Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, Post Office Box 131, Cheongryang, Seoul 130-650, Korea.
3 Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden.
4 Department of Chemistry, The Pennsylvania State University, 104 Chemistry Building, University Park, PA 16802, USA.

* Present address: Nanormics Inc., Seoul, Korea.

{dagger} To whom correspondence should be addressed. Email: hskim76{at}kaist.ac.kr

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THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease.
K. Repanas, N. Zingler, L. E. Layer, G. G. Schumann, A. Perrakis, and O. Weichenrieder (2007)
Nucleic Acids Res.
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Science. ISSN 0036-8075 (print), 1095-9203 (online)