Bioconversion of deoxypodophyllotoxin into epipodophyllotoxin in E. coli using human cytochrome P450 3A4
Biotransformation of deoxypodophyllotoxin to epipodophyllotoxin (ePTOX) by three major human hepatic enzymes, CYP1A2, CYP2C9 and CYP3A4, heterologously expressed in E. coli DH5α, was investigated. It was shown that CYP3A4 catalysed the hydroxylation of deoxypodophyllotoxin into epipodo-phyllotoxin in yields up to 90%. The structure of the metabolite was determined using HPLC-MS and HPLC-SPE-NMR techniques . There was no detectable production of epipodophyllotoxin or podophyllotoxin by CYP1A2 and CYP2C9 enzymes. The CYP3A4 enzyme shows a distinctly different reactivity to deoxypodophyllotoxin compared to the semisynthetic derivatives etoposide and teniposide, which are degraded by 3-O-demethylation. These findings demonstrate a novel system for the production of 2,7'-cyclolignans, starting from the easily accessible deoxypodophyllotoxin. Further kinetic analysis revealed that the Michaelis–Menten Km and Vmax for hydroxylation of deoxypodophyllotoxin by CYP3A4at C-7 position were 1.93µM and 1.48 nmol/min/nmol, respectively. Deoxypodophyllotoxin was subjected to automated docking analysis in order to get better knowledge of the interaction between the CYP 3A4 enzyme and the substrate, using the PatchDock algorithm with distance constraints. Automated docking showed that the β-hydrogen atom at C-7 position is in the most appropriate binding orientation at the site of oxidation. The docking results are consistent with the experimental data for the bioconversion of deoxypodophyllotoxin into epipodophyllotoxin by CYP 3A4 . In addition, the effects of five lignans, deoxypodophyllotoxin, epipodophyllotoxin, podophyllotoxin, demethylenedeoxyodophyllotoxin, and demethylenepodo-phyllotoxin, on CYP3A4 were compared in order to investigate the influence of the methylenedioxy group on the biotransformation process. The docking method hypothesized inhibition by epipodophyllotoxin when the methylenedioxy moiety is involved and the substrate is oriented towards the iron atom of the heme group of CYP 3A4.
Acknowledgements: Van Huygens Short-Term Fellowship is gratefully acknowledged for the financial support to N.P. Vasilev.
References: 1. Vasilev, N.P. et al. (2006) Journal of Biotechnology 126:383–393.
2. Julsing, M.K. et al. (2008) European Journal of Medicinal Chemistry (in press)