Expanding the range of substrate acceptance of enzymes: Combinatorial active-site saturation test

Angewandte Chemie - International Edition

Volumn 44, Issue 27, 2005, Pages 4192-4196

  Reetz, Manfred T ^a   Bocola, Marco ^a   Carballeira, José Daniel ^a   Zha, Dongxing ^a   Vogel, Andreas ^a  

^a MAX PLANCK INSTITUT FÜR KOHLENFORSCHUNG   (Germany)

Author keywords

Directed evolution; Enzymes; Lipases; Protein engineering; Substrate acceptance

Indexed keywords

AMINO ACIDS; SATURATION (MATERIALS COMPOSITION); SUBSTRATES;

COMBINATORIAL ACTIVE-SITE SATURATION TEST (CAST); MUTANTS; RANDOMIZATION; SATURATION TESTS;

ENZYMES;

TRIACYLGLYCEROL LIPASE;

ARTICLE; BINDING SITE; CHEMICAL STRUCTURE; CHEMISTRY; ENZYME SPECIFICITY; ENZYMOLOGY; GENETICS; HYDROLYSIS; METABOLISM; MUTATION; PROTEIN DATABASE; PROTEIN SECONDARY STRUCTURE; PROTEIN TERTIARY STRUCTURE; PSEUDOMONAS AERUGINOSA;

BINDING SITES; DATABASES, PROTEIN; HYDROLYSIS; LIPASE; MODELS, MOLECULAR; MUTATION; PROTEIN STRUCTURE, SECONDARY; PROTEIN STRUCTURE, TERTIARY; PSEUDOMONAS AERUGINOSA; SUBSTRATE SPECIFICITY;

EID: 22144485602     PISSN: 14337851     EISSN: None     Source Type: Journal    
DOI: 10.1002/anie.200500767     Document Type: Article

References (69)

1. a) K. A. Powell, S. W. Ramer, S. B. del Cardayré, W. P. C. Stemmer, M. B. Tobin, P. F. Longchamp, G. W. Huisman, Angew. Chem. 2001, 113, 4068-4080;
2. Angew. Chem. Int. Ed. 2001, 40, 3948-3959;
3. b) F. H. Arnold, G. Georgiou, Directed Enzyme Evolution: Screening and Selection Methods, Vol. 230, Humana Press, Totowa, NJ, 2003;
4. c) S. Brakmann, K. Johnsson, Directed Molecular Evolution of Proteins (or How to Improve Enzymes for Biocatalysis), Wiley-VCH, Weinheim, 2002;
5. d) A. Svendsen, Enzyme Functionality - Design, Engineering, and Screening, Marcel Dekker, New York, 2004;
6. e) S. Brakmann, A. Schwienhorst, Evolutionary Methods in Biotechnology (Clever Tricks for Directed Evolution), Wiley-VCH, Weinheim, 2004.
7. a) M. T. Reetz, Angew. Chem. 2001, 113, 292-320;
8. Angew. Chem. Int. Ed. 2001, 40, 284-310;
9. b) M. T. Reetz in reference [1d], pp. 559-598;
10. c) D. Wahler, J.-L. Reymond, Curr. Opin. Biotechnol. 2001, 12, 535-544.
11. Review: F. Cedrone, A. Menez, E. Quemeneur, Curr. Opin. Struct. Biol. 2000, 10, 405-410.
12. a) M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E. Jaeger, Angew. Chem. 1997, 109, 2961-2963;
13. Angew. Chem. Int. Ed. Engl. 1997, 36, 2830-2832;
14. b) M. T. Reetz, S. Wilensek, D. Zha, K.-E. Jaeger, Angew. Chem. 2001, 113, 3701-3703;
15. Angew. Chem. Int. Ed. 2001, 40, 3589-3591;
16. c) M. T. Reetz, Tetrahedron 2002, 58, 6595-6602;
17. d) M. Bocola, N. Otte, K.-E. Jaeger, M. T. Reetz, W. Thiel, ChemBioChem 2004, 5, 214-223:
18. e) M. T. Reetz, Proc. Natl. Acad. Sci. USA 2004, 101, 5716-5722.
19. a) M. T. Reetz, C. Torre, A. Eipper, R. Lohmer, M. Hermes, B. Brunner, A. Maichele, M. Bocola, M. Arand, A. Cronin, Y. Genzel, A. Archelas, R. Furstoss, Org. Lett. 2004, 6, 177-180;
20. b) F. Cedrone, S. Niel, S. Roca, T. Bhatnagar, N. Ait-Abdelkader, C. Torre, H. Krumm, A. Maichele, M. T. Reetz, J. C. Baratti, Biocatal. Biotransform. 2003, 21, 357-364.
21. a) M. T. Reetz, B. Brunner, T. Schneider, F. Schulz, C. M. Clouthier, M. M. Kayser, Angew. Chem. 2004, 116, 4167-4170;
22. Angew. Chem. Int. Ed. 2004, 43, 4075-4078;
23. b) M. T. Reetz, F. Daligault, B. Brunner, H. Hinrichs, A. Deege, Angew. Chem. 2004, 116, 4170-4173;
24. Angew. Chem. Int. Ed. 2004, 43, 4078-4081.
25. Reviews: references [4c, e]; "Changing the Enantioselectivity of Enzymes by Directed Evolution": M. T. Reetz, Methods Enzymol. 2004, 388, 238-256;
26. N. J. Turner, Trends Biotechnol. 2003, 21, 474-478.
27. a) S. Schlehuber, A. Skerra, Biophys. Chem. 2002, 96, 213-228;
28. b) A. Knappik, L. Ge, A. Honegger, P. Pack, M. Fischer, G. Wellnhofer, A. Hoess, J. Wölle, A. Plückthun, B. Virnekäs, J. Mol. Biol. 2000, 296, 57-86.
29. Examples: a) L. B. Evnin, J. R. Vásquez, C. S. Craik, Proc. Natl. Acad. Sci. USA 1990, 87, 6659-6663;
30. b) J. Yang, Y. Koga, H. Nakano, T. Yamane, Protein Eng. 2002, 15, 147-152;
31. c) E. Whittle, J. Shanklin, J. Biol. Chem. 2001, 276, 21500-21505;
32. d) A. R. Schmitzer, F. Lépine, J. N. Pelletier, Protein Eng. Des. Sel. 2004, 17, 809-819;
33. e) C. M. Hill, W.-S. Li, J. B. Thoden, H. M. Holden, F. M. Raushel, J. Am. Chem. Soc. 2003, 125, 8990-8991;
34. f) F. Brühlmann, W. Chen, Biotechnol. Bioeng. 1999, 63, 544-551;
35. g) E. M. Gabor, D. B. Janssen, Protein Eng. Des. Sel. 2004, 17, 571-579;
36. h) J. Yang, Y. Koga, H. Nakano, T. Yamane, Protein Eng. 2002, 15, 147-152;
37. i) S. V. Taylor, P. Kast, D. Hilvert, Angew. Chem. 2001, 113, 3408-3436;
38. Angew. Chem. Int. Ed. 2001, 40, 3310-3335.
39. a) M. Chen-Goodspeed, M. A. Sogorb, F. Wu, S.-B. Hong, F. M. Raushel, Biochemistry 2001, 40, 1325-1331;
40. b) "Directed Evolution of Enantioselective Enzymes as Catalysts in the Production of Chiral Pharmaceuticals": M. T. Reetz in Pharmacochemistry Library, Vol. 32 (Trends in Drug Research III) (Ed.: H. van der Goot), Elsevier, Amsterdam, 2002, pp. 27-37;
41. c) Y. Koga, K. Kato, H. Nakano, T. Yamane, J. Mol. Biol. 2003, 331, 585-592;
42. d) S. Park, K. L. Morley, G. P. Horsman, M. Holmquist, K. Hult, R. J. Kazlauskas, Chem. Biol. 2005, 12, 45-54.
43. a) M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M. Keßeler, R. Stürmer, T. Zelinski, Angew. Chem. 2004, 116, 806-843;
44. Angew. Chem. Int. Ed. 2004, 43, 788-824;
45. b) K. Drauz, H. Waldmann, Enzyme Catalysis in Organic Synthesis: A Comprehensive Handbook, Vol. I-III, 2nd ed., Wiley-VCH, Weinheim, 2002;
46. c) K. Faber, Biotransformations in Organic Chemistry, 4th ed., Springer, Berlin, 2000;
47. d) A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, B. Witholt, Nature 2001, 409, 258-268.
48. Discussions of enzyme promiscuity, plasticity, and so-called moonlighting: a) A. Aharoni, L. Gaidukov, O. Khersonsky, S. McQ Gould, C. Roodveldt, D. S. Tawfik, Nat. Genet. 2005, 37, 73-76;
49. b) S. Raillard, A. Krebber, Y. Chen, J. E. Ness, E. Bermudez, R. Trinidad, R. Fullem, C. Davis, M. Welch, J. Seffernick, L. P. Wackett, W. P. C. Stemmer, J. Minshull, Chem. Biol. 2001, 8, 891-898;
50. c) P. J. O'Brien, D. Herschlag, Chem. Biol. 1999, 6, R91-R105;
51. d) S. D. Copley, Curr. Opin. Chem. Biol. 2003, 7, 265-272;
52. e) U. T. Bornscheuer, R. J. Kazlauskas, Angew. Chem. 2004, 116, 6156-6165;
53. Angew. Chem. Int. Ed. 2004, 43, 6032-6040.
54. The randomization at two amino acids which are not spatially close to each other yet have side chains pointing toward the active site (for example, in two different libraries) may of course also be considered.
55. a) A. Fersht, Structure and Mechanism in Protein Science, W. H. Freeman, New York, 1999;
56. b) R. A. Copeland, Enzymes, Wiley-VCH, Weinheim, 2000.
57. These guidelines are derived from ideal secondary structural units. If in a given case the latter are not ideal, as, for example, in a kinked helix, then additional positions need to be considered. In the case of a kinked helix, for example, it may be a sequentially neighboring amino acid (n+1). It is mandatory to include such structural aspects when designing combinatorial active-site saturation test libraries, that is, the shape of the binding pocket serves as a guide.
58. Examples: a) G. DeSantis, K. Wong, B. Farwell, K. Chatman, Z. Zhu, G. Tomlinson, H. Huang, X. Tan, L. Bibbs, P. Chen, K. Kretz, M. J. Burk, J. Am. Chem. Soc. 2003, 125, 11476-11477;
59. b) S. A. Funke, A. Eipper, M. T. Reetz, N. Otte, W. Thiel, G. van Pouderoyen, B. W. Dijkstra, K.-E. Jaeger, T. Eggert, Biocatal. Biotransform. 2003, 21, 67-73;
60. c) R. Georgescu, G. Bandara, L. Sun in Directed Evolution Library Creation (Eds.: F. H. Arnold, G. Georgiou), Humana Press, Totowa, NJ, 2003, pp. 75-89.
61. For this calculation we used the traditional statistical method; see, for example: M. S. Warren, S. J. Benkovic, Protein Eng. 1997, 10, 63-68, and references therein.
62. a) K.-E. Jaeger, B. Schneidinger, K. Liebeton, D. Haas, M. T. Reetz, S. Philippou, G. Gerritse, S. Ransac, B. W. Dijkstra in Molecular Biology and Biotechnological Application (Eds.: T. Nakazawa, K. Furukawa, D. Haas, S. Silver), ASM Press, Washington, 1996, pp. 319-339;
63. b) K.-E. Jaeger, K. Liebeton, A. Zonta, K. Schimossek, M. T. Reetz, Appl. Microbiol. Biotechnol. 1996, 46, 99-105.
64. We thank K.-E. Jaeger for providing the lipase-expression system.[18]
65. M. Nardini, D. A. Lang, K. Liebeton, K.-E. Jaeger, B. W. Dijkstra, J. Biol. Chem. 2000, 275, 31219-31225.
66. Because of the instability of the lipase from Pseudomonas aeruginosa,[18] it was not possible to purify mutants and to perform detailed kinetic studies. A previous kinetic study was performed by using the supernatants and assuming similar amounts of enzyme expressed and secreted (M. T. Reetz, S. Wilensek, unpublished results;
67. see also: M. T. Reetz, Tetrahedron 2002, 58, 6595-6602). To lend support to this assumption, we measured the lipase activity towards the "natural" compound palmitic acid p-nitrophenyl ester as a reference substrate. All of the mutants of the present study showed similar activities towards this reference substrate but slightly lower activities relative to the WT enzyme. Therefore, the low activities of the WT enzyme towards the different substrates in Figure 3 are not because of a lower expression rate. This was confirmed by sodium dodecylsulfate PAGE analysis. This means that the profiles of the mutants reflect inherent activity enhancements.
68. A comparative study encompassing various substrates and lipases is underway, as are CASTing experiments on commercially available lipases that have higher thermal stability than the lipase from Pseudomonas aeruginosa.
69. None of the variants contain additional mutations because of the PCR.