Reshaping an Enzyme Binding Pocket for Enhanced and Inverted Stereoselectivity: Use of Smallest Amino Acid Alphabets in Directed Evolution†
Dr. Zhoutong Sun
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg (Germany)
Search for more papers by this authorDr. Richard Lonsdale
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg (Germany)
Search for more papers by this authorXu-Dong Kong
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)
Search for more papers by this authorProf. Jian-He Xu
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)
Search for more papers by this authorCorresponding Author
Prof. Jiahai Zhou
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032 (China)
Jiahai Zhou, State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032 (China)
Manfred T. Reetz, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
Search for more papers by this authorCorresponding Author
Prof. Dr. Manfred T. Reetz
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg (Germany)
Jiahai Zhou, State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032 (China)
Manfred T. Reetz, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
Search for more papers by this authorDr. Zhoutong Sun
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg (Germany)
Search for more papers by this authorDr. Richard Lonsdale
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg (Germany)
Search for more papers by this authorXu-Dong Kong
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)
Search for more papers by this authorProf. Jian-He Xu
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)
Search for more papers by this authorCorresponding Author
Prof. Jiahai Zhou
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032 (China)
Jiahai Zhou, State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032 (China)
Manfred T. Reetz, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
Search for more papers by this authorCorresponding Author
Prof. Dr. Manfred T. Reetz
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35032 Marburg (Germany)
Jiahai Zhou, State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032 (China)
Manfred T. Reetz, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
Search for more papers by this authorThis research was supported by the Max Planck Society, the LOEWE Research Cluster SynChemBio, and the Arthur C. Cope Fund, in addition to grants from the National Program on Key Basic Research of China (2011CB7710800).
Abstract
Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme.
Supporting Information
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
| Filename | Description |
|---|---|
| ange_201501809_sm_miscellaneous_information.pdf3.8 MB | miscellaneous_information |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- 1For reviews of directed evolution, see:
- 1a Directed Evolution Library Creation: Methods in Molecular Biology (Eds.: ), Humana Press, Totowa, 2014;
- 1bM. T. Reetz, Angew. Chem. Int. Ed. 2011, 50, 138–174; Angew. Chem. 2011, 123, 144–182;
- 1cA. S. Bommarius, J. K. Blum, A. J. Abrahamson, Curr. Opin. Chem. Biol. 2011, 15, 194–200;
- 1dC. Jäckel, D. Hilvert, Curr. Opin. Biotechnol. 2010, 21, 753–759;
- 1eE. M. Brustad, F. H. Arnold, Curr. Opin. Chem. Biol. 2011, 15, 201–210;
- 1fM. Goldsmith, D. S. Tawfik, Curr. Opin. Struct. Biol. 2012, 22, 406–412;
- 1gM. T. Reetz, J. Am. Chem. Soc. 2013, 135, 12480–12496;
- 1hM. Widersten, Curr. Opin. Chem. Biol. 2014, 21, 42–47;
- 1iC. A. Denard, H. Ren, H. Zhao, Curr. Opin. Chem. Biol. 2015, 25, 55–64;
- 1jA. Currin, N. Swainston, P. J. Day, D. B. Kell, Chem. Soc. Rev. 2015, 44, 1172–1239;
- 1kN. J. Turner, Nat. Chem. Biol. 2009, 5, 567–573;
- 1lS. Lutz, U. T. Bornscheuer, Protein Engineering Handbook, Wiley-VCH, Weinheim, 2009.
- 2For a selection of recent applications of ISM, see:
- 2aY. Yang, Y. T. Chi, H. H. Toh, Z. Li, Chem. Commun. 2015, 51, 914;
- 2bK. Koketsu, Y, Shomura, K. Moriwaki, M. Hayashi, S. Mitsuhashi, R. Hara, K. Kino, Y. Higuchi, ACS Synth. Biol. 2015, DOI: 10.1021/sb500247a;
- 2cA. Z. Walton, B. Sullivan, A. Paterson-Orazem, J. D. Stewart, ACS Catal. 2014, 4, 2307–2318;
- 2dG. Li, J. Ren, P. Yao, Y. Duan, H. Zhang, Q. Wu, J. Feng, P. C. K. Lau, D. Zhu, ACS Catal. 2014, 4, 903–908;
- 2eX. Wang, K. Zheng, H. Zheng, H. Nie, Z. Yang, L. Tang, J. Biotechnol. 2014, 192, 102–107;
- 2fC. Chen, J. Van der Borght, R. De Vreese, M. D’hooghe, W. Soetaert, T. Desmet, Chem. Commun. 2014, 50, 7834;
- 2gC. Blikstad, K. M. Dahlström, T. A. Salminen, M. Widersten, FEBS J. 2014, 281, 2387–2398;
- 2hZ.-G. Zhang, R. Lonsdale, J. Sanchis, M. T. Reetz, J. Am. Chem. Soc. 2014, 136, 17262–17272;
- 2iC. Blikstad, K. M. Dahlström, T. A. Salminen, M. Widersten, ACS Catal. 2013, 3, 3016–3025;
- 2jA. Bosshart, S. Panke, M. Bechtold, Angew. Chem. Int. Ed. 2013, 52, 9673–9676; Angew. Chem. 2013, 125, 9855–9858.
- 3
- 3aW. M. Patrick, A. E. Firth, Biomol. Eng. 2005, 22, 105–112;
- 3bA. E. Firth, W. M. Patrick, Nucleic Acids Res. 2008, 36, W 281–285;
- 3cM. Denault, J. N. Pelletier, in Protein Engineering Protocols, Vol. 352 (Eds.: ), Humana Press, Totowa, NJ, 2007, pp. 127–154;
- 3dCASTER computer aid for designing saturation mutagenesis libraries: http://www.kofo.mpg.de/en/research/biocatalysis.
- 4
- 4aM. T. Reetz, S. Wu, Chem. Commun. 2008, 5499–5501;
- 4bA. G. Sandström, Y. Wikmark, K. Engström, J. Nyhlén, J.-E. Bäckvall, Proc. Natl. Acad. Sci. USA 2012, 109, 78–83;
- 4cA. Nobili, M. G. Gall, I. V. Pavlidis, M. L. Thompson, M. Schmidt, U. T. Bornscheuer, FEBS J. 2013, 280, 3084–3093.
- 5
- 5aG. A. Weiss, C. K. Watanabe, A. Zhong, A. Goddard, S. S. Sidhu, Proc. Natl. Acad. Sci. USA 2000, 97, 8950–8954;
- 5bS. S. Sidhu, Protein Sci. 2005, 14, 2405–2413;
- 5cS. S. Sidhu, A. A. Kossiakoff, Curr. Opin. Chem. Biol. 2007, 11, 347–354.
- 6
- 6aK. L. Morrison, G. A. Weiss, Curr. Opin. Chem. Biol. 2001, 5, 302–307;
- 6bI. Massova, P. A. Kollman, J. Am. Chem. Soc. 1999, 121, 8133–8143;
- 6cJ. C. Lewis, S. M. Mantovani, Y. Fu, C. D. Snow, R. S. Komor, C.-H. Wong, F. H. Arnold, ChemBioChem 2010, 11, 2502–2505.
- 7
- 7aM. J. van der Werf, K. M. Overkamp, J. A. de Bont, J. Bacteriol. 1998, 180, 5052–5057;
- 7bM. Arand, B. M. Hallberg, J. Zou, T. Bergfors, F. Oesch, M. J. van der Werf, J. A. M. de Bont, T. A. Jones, S. L. Mowbray, EMBO J. 2003, 22, 2583–2592;
- 7cM. J. van der Werf, R. V. A. Orru, K. M. Overkamp, H. J. Swarts, I. Osprian, A. J. Steinreiber, A. M. de Bont, K. Faber, Appl. Microbiol. Biotechnol. 1999, 52, 380–385.
- 8H. Zheng, M. T. Reetz, J. Am. Chem. Soc. 2010, 132, 15744–15751.
- 9
- 9aD. Wahler, J. L. Reymond, Angew. Chem. Int. Ed. 2002, 41, 1229–1232;
10.1002/1521-3773(20020402)41:7<1229::AID-ANIE1229>3.0.CO;2-5 CAS PubMed Web of Science® Google ScholarAngew. Chem. 2002, 114, 1277–1280;
- 9bJ. L. Reymond, Enzyme Assays: High-Throughput Screening, Genetic Selection and Fingerprinting, Wiley-VCH, Weinheim, 2006.
- 10For a review of additive versus non-additive mutational effects in protein engineering, see: M. T. Reetz, Angew. Chem. Int. Ed. 2013, 52, 2658–2666; Angew. Chem. 2013, 125, 2720–2729.
- 11
- 11aM. Wada, C. C. Hsu, D. Franke, M. Mitchell, A. Heine, I. Wilson, C.-H. Wong, Bioorg. Med. Chem. 2003, 11, 2091–2098;
- 11bM. T. Reetz, M. Bocola, L.-W. Wang, J. Sanchis, A. Cronin, M. Arand, J. Zou, A. Archelas, A.-L. Bottalla, A. Naworyta, S. L. Mowbray, J. Am. Chem. Soc. 2009, 131, 7334–7343.
- 12
- 12aK. H. Hopmann, B. M. Hallberg, F. Himo, J. Am. Chem. Soc. 2005, 127, 14339–14347;
- 12bM. E. S. Lind, F. Himo, Angew. Chem. Int. Ed. 2013, 52, 4563–4567; Angew. Chem. 2013, 125, 4661–4665.
- 13Q. Q. Hou, X. Sheng, J. H. Wang, Y. J. Liu, C. B. Liu, Biochim. Biophys. Acta Proteins Proteomics 2012, 1824, 263–268.
- 14J. Chandrasekhar, S. F. Smith, W. L. Jorgensen, J. Am. Chem. Soc. 1985, 107, 154–163.
- 15
- 15aG. Stork, L. D. Cama, D. R. Coulson, J. Am. Chem. Soc. 1974, 96, 5268–5270;
- 15bJ. Na, K. N. Houk, C. G. Shevlin, K. D. Janda, R. A. Lerner, J. Am. Chem. Soc. 1993, 115, 8453–8454;
- 15cT. Laitinen, J. Rouvinen, M. Peräkylä, J. Org. Chem. 1998, 63, 8157–8162.
- 16Amino Acid Properties and Consequences of Substitutions: M. J. Betts, R. B. Russell in Bioinformatics for Geneticists (Eds. ), Wiley, New York, 2003.
- 17aJ. Lameira, B. Ram Prasad, Z. T. Chu, A. Warshel, Proteins 2015, 83, 318–330;
- 17bA. Warshel, P. K. Sharma, M. Kato, Y. Xiang, H. Liu, M. H. M. Olsson, Chem. Rev. 2006, 106, 3210–3235;
- 17cA. Warshel, Angew. Chem. Int. Ed. 2014, 53, 10020–10031; Angew. Chem. 2014, 126, 10182–10194.
Citing Literature
This is the
German version
of Angewandte Chemie.
Note for articles published since 1962:
Do not cite this version alone.
Take me to the International Edition version with citable page numbers, DOI, and citation export.
We apologize for the inconvenience.
