Rapid characterization of the major drug-metabolizing human hepatic cytochrome P-450 enzymes expressed in Escherichia coli

The major drug-metabolizing human hepatic cytochrome P-450s (CYPs; CYP1A2, 2C9, 2C19, 2D6, and 3A4) coexpressed functionally in Escherichia coli with human NADPH-P-450 reductase have been validated as surrogates to their coun terparts in human liver microsomes (HLM) using automated technology. The de alkylation of ethoxyresorufin, dextromethorphan, and erythromycin were all shown to be specific reactions for CYP1A2, CYP2D6, and CYP3A4 that allowed direct comparison with kinetic data for HLM. For CYP2C9 and CYP2C19, the ki netics for the discrete oxidations of naproxen and diazepam were compared t o data obtained using established, commercial CYP preparations. Turnover nu mbers of CYPs expressed in E. coli toward these substrates were generally e qual to or even greater than those of the major commercial suppliers [CYP1A 2 (ethoxyresorufin), E. coli 0.6 +/- 0.2 min(-1) versus B lymphoblasts 0.4 +/- 0.1 min(-1); CYP2C9 (naproxen), 6.7 +/- 0.9 versus 4.9 min(-1); CYP2C19 (diazepam), 3.7 +/- 0.3 versus 0.2 +/- 0.1 min(-1); CYP2D6 (dextromethorph an), 4.7 +/-: 0.1 versus 4.4 +/- 0.1 min(-1); CYP3A4 (erythromycin), 3 +/- 1.2 versus 1.6 min(-1)]. The apparent K-m values for the specific reactions were also similar (K-m ranges for expressed CYPs and HLM were: ethoxyresor ufin 0.5-1.0 mu M, dextromethorphan 1.3-5.9 mu M, and erythromycin 18-57 mu M)I indicating little if any effect of N-terminal modification on the E. c oli-expressed CYPs. The data generated for all the probe substrates by HLM and recombinant CYPs also agreed well with literature values. In summary, E . coli-expressed CYPs appear faithful surrogates for the native (HLM) enzym e, and these data suggest that such recombinant enzymes may be suitable for predictive human metabolism studies.