Abstract
Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.
Footnotes
This work was supported in part by the 21C Frontier Microbial Genomics and the Application Center Program of the Ministry of Education, Science and Technology of the Republic of Korea; the Korea Science and Engineering Foundation [Grant R01-2008-000-21072-02008]; and the Second Stage BK21 Project from the Ministry of Education, Science and Technology of the Republic of Korea.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.030759.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- P450
- cytochrome P450
- CYP102A1
- P450 BM3
- PCR
- polymerase chain reaction
- 7-OH coumarin
- 7-hydroxycoumarin
- 3-OH coumarin
- 3-hydroxycoumarin
- LB
- Luria-Bertani
- IPTG
- isopropyl-β-d-thiogalactopyranoside
- PhOD
- phenacetin O-deethylation
- EROD
- 7-ethoxyresorufin O-deethylation
- HPLC
- high-performance liquid chromatography
- LC/MS/MS
- liquid chromatography-tandem mass spectrometry
- MD
- molecular dynamics
- Tm
- thermal stability.
- Received October 14, 2009.
- Accepted January 25, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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