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PEDS Advance Access originally published online on October 18, 2006
Protein Engineering Design and Selection 2006 19(12):547-554; doi:10.1093/protein/gzl042
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide

Santosh Kumar1,3,4, Ling Sun1,3, Hong Liu2, B.K. Muralidhara1 and James R. Halpert1

1 Department of Pharmacology and Toxicology, University of Texas Medical Branch 301 University Boulevard, Galveston, TX 77555-1031, USA 2 Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Drug Discovery and Design Center 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Pudong, Shanghai 201203, P.R. China

4To whom correspondence should be addressed. Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1031, USA E-mail: sakumar{at}utmb.edu

The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H2O2-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in Km,HOOH for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening >3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35–55°C) and maintained ~1.4-fold higher activity than QM at 45°C for 6 h. In addition, QM/L295H showed a significant increase in Tm,app compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5–15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a >2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent.

Keywords: Cytochrome P450/directed evolution/heme accessibility/random mutagenesis/thermostability

Received July 24, 2006; revised August 21, 2006; accepted August 23, 2006.


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