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Ligand Diversity of Human and Chimpanzee CYP3A4: Activation of Human CYP3A4 by Lithocholic Acid Results from Positive Selection

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California-San Diego, La Jolla, California (S.K., J.R.H.); Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri (S.K.); Department of Pharmacology, Johannes Gutenberg University Mainz, Mainz, Germany (H.Q., L.W.); Department of Biochemistry and Molecular Biology and the Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas (N.O.); and Institute of Anthropology, Johannes Gutenberg University Mainz, Mainz, Germany (H.H.)

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 -naphthoflavone (25 µM), human CYP3A4 showed a slightly decreased substrate concentration at which 50% of the maximal rate V_max 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.