Abstract
Recently, the wild-type and mutant forms of cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium were found to oxidize various xenobiotic substrates, including pharmaceuticals, of human P450 enzymes. Simvastatin and lovastatin, which are used to treat hyperlipidemia and hypercholesterolemia, are oxidized by human CYP3A4/5 to produce several metabolites, including 6′β-hydroxy (OH), 3″-OH, and exomethylene products. In this report, we show that the oxidation of simvastatin and lovastatin was catalyzed by wild-type CYP102A1 and a set of its mutants, which were generated by site-directed and random mutagenesis. One major hydroxylated product (6′β-OH) and one minor product (6′-exomethylene), but not other products, were produced by CYP102A1 mutants. Formation of the metabolites was confirmed by high-performance liquid chromatography, liquid chromatography-mass spectroscopy, and NMR. Chemical methods to synthesize the metabolites of simvastatin and lovastatin have not been reported. These results demonstrate that CYP102A1 mutants can be used to produce human metabolites, especially chiral metabolites, of simvastatin and lovastatin. Our computational findings suggest that a conformational change in the cavity of the mutant active sites is related to the activity change. The modeling results also suggest that the activity change results from the movement of several specific residues in the active sites of the mutants. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward simvastatin and lovastatin.
Footnotes
- Received September 17, 2010.
- Accepted October 20, 2010.
K.-H.K., J.-Y.K., and D.-H.K. contributed equally to this work.
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 National Research Foundation [Grant 2010-0027685]; the Second Stage BK21 Project from the Ministry of Education, Science and Technology of the Republic of Korea; and the Support Program for the Advancement of National Research Facilities and Equipment of the Ministry of Education, Science and Technology of the Republic of Korea (2010).
Parts of this work were previously presented at the following conference: Kang JY, Kim KH, Kim DH, Jung HC, Pan JG, Ahn T, and Yun CH (2009) Generation of human metabolites of simvastatin and lovastatin by bacterial cytochrome P450 BM3 enzymes [abstract]. Drug Metab Rev 41 (Suppl 3):47. 16th North American Regional ISSX Meeting; 2009 Oct 18–22; Baltimore, MD. International Society for the Study of Xenobiotics, Washington, DC.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.036392.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.ABBREVIATIONS:
- OH
- hydroxy
- FDA
- U.S. Food and Drug Administration
- MIST
- metabolites in safety testing
- WT
- wild type
- P450
- cytochrome P450
- CYP102A1
- cytochrome P450 BM3
- HPLC
- high-performance liquid chromatography
- LC
- liquid chromatography
- MS
- mass spectrometry
- αNF
- 7,8-benzoflavone
- LOV
- lovastatin
- SIM
- simvastatin
- ESP
- electrostatic potential
- MM
- molecular mechanics
- PBSA
- Poisson Boltzmann and surface area
- MD
- molecular dynamics.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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Generation of Human Chiral Metabolites of Simvastatin and Lovastatin by Bacterial CYP102A1 Mutants
