Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

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Abstract

Based on the X-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)- and bifonazole (BIF)-bound P450 2B4, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a > 4-fold decrease in differential inhibition by BIF and 4-CPI (IC50,BIF/IC50,4-CPI). F296A, T302A, I363A, V367A, and V477A showed a ⩾2-fold decreased kcat for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered Km. T302A, V367L, and V477A showed > 4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered stereo- and regioselectivity. Interestingly, I363A showed a ⩾150-fold enhanced kcat/Km with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis.

Section snippets

Materials

7-EFC and 7-hydroxy-4-trifluoromethylcoumarin (7-HFC) were purchased from Molecular Probes Inc. (Eugene, OR). NADPH was from Sigma Chemical Co. (St. Louis, MO). [4-14C]-testosterone was obtained from Amersham Biosciences (Piscataway, NJ). Authentic steroid standards were obtained from Steraloids Inc. (Newport, RI). Oligonucleotide primers were obtained from Sigma Genosys (Woodlands, TX). The QuikChange site-directed mutagenesis kit was obtained from Stratagene (La Jolla, CA). The GeneClean kit

Inhibition studies with 4-CPI and BIF

To examine the effect of the mutations on inhibitor binding, P450 2B4dH/H226Y and site-directed mutants were analyzed for 4-CPI and BIF inhibition using 7-EFC O-deethylation. The results are presented in Table 2 and Fig. 1. Because of the dominance of the strong coordinate bond between the heme iron of P450 and nitrogen of the imidazole ring, phenylimidazole compounds show non-competitive inhibition, such that the IC50 value is comparable to the Ki value. Furthermore, inhibition studies with

Conclusions

This is the first study to examine the utility of the ligand-bound P450 2B4dH/H226Y X-ray crystal structures for predicting differential inhibition by BIF and 4-CPI as well as substrate turnover and stereo- and regioselectivity. The study was carried out by substituting amino acid residues that interact with 4-CPI-, BIF-, or both with smaller and/or larger ones followed by characterization of enzyme inhibition and catalysis. Interestingly, T302A, I363A, V367A, and V477A, which were created

Acknowledgments

The authors thank Ms. Ling Sun for her technical assistance. We also thank Drs. Harshica Fernando, B.K. Muralidhara, and YongHong Zhao for their expert suggestions. Financial support was provided by NIH Grant ES03619 and Center Grant ES06676 (to J.R.H.).

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