Establishing wild-type levels of catalytic activity on natural and artificial (βα)8-barrel protein scaffolds
- aInstitute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany;
- bMax Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tübingen, Germany; and
- cCenter for Bioinformatics, Division for Simulation of Biological Systems, Eberhard Karls University Tübingen, Sand 14, D-72076 Tübingen, Germany
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Edited by Frances H. Arnold, California Institute of Technology, Pasadena, CA, and approved January 6, 2009 (received for review October 14, 2008)
Abstract
The generation of high levels of new catalytic activities on natural and artificial protein scaffolds is a major goal of enzyme engineering. Here, we used random mutagenesis and selection in vivo to establish a sugar isomerisation reaction on both a natural (βα)8-barrel enzyme and a catalytically inert chimeric (βα)8-barrel scaffold, which was generated by the recombination of 2 (βα)4-half barrels. The best evolved variants show turnover numbers and substrate affinities that are similar to those of wild-type enzymes catalyzing the same reaction. The determination of the crystal structure of the most proficient variant allowed us to model the substrate sugar in the novel active site and to elucidate the mechanistic basis of the newly established activity. The results demonstrate that natural and inert artificial protein scaffolds can be converted into highly proficient enzymes in the laboratory, and provide insights into the mechanisms of enzyme evolution.
Footnotes
- 1To whom correspondence should be addressed. E-mail: reinhard.sterner{at}biologie.uni-regensburg.de
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Author contributions: B.H. and R.S. designed research; J.C. and B.H. performed research; J.C., C.M., B.H., and R.S. analyzed data; and B.H. and R.S. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID code 2w79).
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This article contains supporting information online at www.pnas.org/cgi/content/full/0810342106/DCSupplemental.
