Abstract
For currently unknown reasons, the evolution of CYP3A4 underwent acceleration in the human lineage after the split from chimpanzee. We investigated the significance of this event by comparing Escherichia coli-expressed CYP3A4 from humans, chimpanzee, and their most recent common ancestor. The expression level of chimpanzee CYP3A4 was ∼50% of the human CYP3A4, whereas ancestral CYP3A4 did not express in E. coli. Steady-state kinetic analysis with 7-benzyloxyquinoline, 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and testosterone showed no significant differences between human and chimpanzee CYP3A4. Upon addition of α-naphthoflavone (25 μM), human CYP3A4 showed a slightly decreased substrate concentration at which 50% of the maximal rate Vmax is reached for 7-BFC, whereas chimpanzee CYP3A4 showed a >2-fold increase. No significant differences in inhibition/activation were found for a panel of 43 drugs and endogenous compounds, suggesting that the wide substrate spectrum of human CYP3A4 precedes the human-chimpanzee split. A striking exception was the hepatotoxic secondary bile acid lithocholic acid, which at saturation caused a 5-fold increase in 7-BFC debenzylation by human CYP3A4 but not by chimpanzee CYP3A4. Mutagenesis of human CYP3A4 revealed that at least four of the six amino acids positively selected in the human lineage contribute to the activating effect of lithocholic acid. In summary, the wide functional conservation between chimpanzee and human CYP3A4 raises the prospect that phylogenetically more distant primate species such as rhesus and squirrel monkey represent suitable models of the human counterpart. Positive selection on the human CYP3A4 may have been triggered by an increased load of dietary steroids, which led to a novel defense mechanism against cholestasis.
Footnotes
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This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM54995] (to J.R.H.); and Deutsche Forschungsgemeinschaft [Grant WO505/2-2] (to L.W.).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.024372.
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ABBREVIATIONS: P450, cytochrome P450; 7-BQ, 7-benzyloxyquinoline; 7-BFC, 7-benzyloxy-4-(trifluoromethyl)coumarin; ANF, α-naphthoflavone; PCR, polymerase chain reaction; S50, substrate concentration at which 50% of the maximal rate Vmax is reached; LCA, lithocholic acid.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
- Received September 3, 2008.
- Accepted March 16, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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