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Multiple CH/π Interactions Maintain the Binding of Aflatoxin B₁ in the Active Cavity of Human Cytochrome P450 1A2.

Jun WuSisi ZhuYunbo WuTianqing JiangLingling WangJun JiangJikai WenYiqun Deng
Published in: Toxins (2019)
Human cytochrome P450 1A2 (CYP1A2) is one of the key CYPs that activate aflatoxin B₁ (AFB₁), 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 AFB₁ has not been fully clarified. In this study, we used calculation biology to predict a model of CYP1A2 with AFB₁, 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 AFB₁, while F125A, F226A, and F260A were significantly reduced in their ability to activate AFB₁, implying that hydrogen bonds between Thr-124 and AFB₁ 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 AFB₁ collectively maintained the stable binding of AFB₁ in the active cavity of CYP1A2.
Keyphrases
  • escherichia coli
  • endothelial cells
  • high resolution
  • dna binding
  • room temperature
  • high throughput
  • pseudomonas aeruginosa
  • single cell
  • klebsiella pneumoniae
  • amino acid