Abstract
Human cytochrome P450 1A2 (CYP1A2) is one of the key CYPs that activate aflatoxin B&sub1; (AFB&sub1;), a notorious mycotoxin, into carcinogenic exo-8,9-epoxides (AFBO) in the liver. Although the structure of CYP1A2 is available, the mechanism of CYP1A2-specific binding to AFB&sub1; has not been fully clarified. In this study, we used calculation biology to predict a model of CYP1A2 with AFB&sub1;, where Thr-124, Phe-125, Phe-226, and Phe-260 possibly participate in the specific binding. Site-directed mutagenesis was performed to construct mutants T124A, F125A, F226A, and F260A. Escherichia coli-expressed recombinant proteins T124A, F226A, and F260A had active structures, while F125A did not. This was evidenced by Fe2+∙Carbon monoxide (CO)-reduced difference spectra and circular dichroism spectroscopy. Mutant F125A was expressed in HEK293T cells. Steady kinetic assays showed that T124A had enhanced activity towards AFB&sub1;, while F125A, F226A, and F260A were significantly reduced in their ability to activate AFB&sub1;, implying that hydrogen bonds between Thr-124 and AFB&sub1; were not important for substrate-specific binding, whereas Phe-125, Phe-226, and Phe-260 were essential for the process. The computation simulation and experimental results showed that the three key CH/π interactions between Phe-125, Phe-226, or Phe-260 and AFB&sub1; collectively maintained the stable binding of AFB&sub1; in the active cavity of CYP1A2.