Cirino, Patrick C. and Tang, Yi and Takahashi, Katsuyuki and Tirrell, David A. and Arnold, Frances H. (2003) Global incorporation of norleucine in place of methionine in cytochrome P450 BM-3 heme domain increases peroxygenase activity. Biotechnology and Bioengineering, 83 (6). pp. 729-734. ISSN 0006-3592. doi:10.1002/bit.10718. https://resolver.caltech.edu/CaltechAUTHORS:CIRbb2003
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Abstract
In this study we have replaced all 13 methionine residues in the cytochrome P450 BM-3 heme domain (463 amino acids) with the isosteric methionine analog norleucine. This experiment has provided a means of testing the functional limits of globally incorporating into an enzyme an unnatural amino acid in place of its natural analog, and also an efficient way to test whether inactivation during peroxide-driven P450 catalysis involves methionine oxidation. Although there was no increase in the stability of the P450 under standard reaction conditions (in 10 mM hydrogen peroxide), complete substitution with norleucine resulted in nearly two-fold-increased peroxygenase activity. Thermostability was significantly reduced. The fact that the enzyme can tolerate such extensive amino acid replacement suggests that we can engineer enzymes with unique chemical properties via incorporation of unnatural amino acids while retaining or improving catalytic properties. This system also provides a platform for directing enzyme evolution using an extended set of protein building blocks.
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| Additional Information: | © 2003 Wiley. Received 17 December 2002; accepted 24 March 2003. Published online 23 June 2003. The authors thank Dr. Mona Shahgholi for assistance with the MALDI-TOF mass spectral analyses and the Yale E. coli Genetic Stock Center for providing the methionine auxotroph. Contract grant sponsors: Biotechnology Research and Development Corporation (Peoria, IL); NSF Graduate Research Fellowship; NIH Contract grant number: R01-GM62523-02 | |||||||||
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| Subject Keywords: | protein engineering; norleucine;methionine oxidation; directed evolution; cytochrome P450; peroxygenase | |||||||||
| Issue or Number: | 6 | |||||||||
| DOI: | 10.1002/bit.10718 | |||||||||
| Record Number: | CaltechAUTHORS:CIRbb2003 | |||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:CIRbb2003 | |||||||||
| Official Citation: | Cirino, P. C., Tang, Y., Takahashi, K., Tirrell, D. A. and Arnold, F. H. (2003), Global incorporation of norleucine in place of methionine in cytochrome P450 BM-3 heme domain increases peroxygenase activity. Biotechnol. Bioeng., 83: 729–734. doi: 10.1002/bit.10718 | |||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | |||||||||
| ID Code: | 53314 | |||||||||
| Collection: | CaltechAUTHORS | |||||||||
| Deposited By: | Anne Hormann | |||||||||
| Deposited On: | 07 Feb 2015 19:39 | |||||||||
| Last Modified: | 10 Nov 2021 20:00 |
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