1. We investigated the total metabolism of coumarin by baculovirus (BV)-expressed CYP2A13 and compared it with metabolism by BV-expressed CYP2A6. The major coumarin metabolite formed by CYP2A13 was 7-hydroxycoumarin, which accounted for 43% of the total metabolism. The product of 3,4-epoxidation, o-hydroxyphenylacetaldehyde (o-HPA), accounted for 30% of the total metabolites. 2. The Km and Vmax for CYP2A13-mediated coumarin 7-hydroxylation were 0.48 ± 0.07 μM and 0.15 ± 0.006 nmol min-1 nmol-1 CYP, respectively. The Vmax of coumarin 7-hydroxylation by CYP2A13 was about 16-fold lower than that of CYP2A6, whereas the Km was 10-fold lower. 3. In the mouse, there were two orthologues for CYP2A6: CYP2A4 and CYP2A5, which differed by only 11 amino acids. However, CYP2A5 is an efficient coumarin 7-hydroxylase, where as CYP2A4 is not. We report here that BV-expressed CYP2A4 metabolizes coumarin by 3,4-epoxidation. Two products of the 3,4-epoxidation pathway, o-HPA and o-hydroxyphenylacetic acid (o-HPAA), were detected by radioflow HPLC. 4. The Km and Vmax for the coumarin 3,4-epoxidation by CYP2A4 were 8.7 ± 3.6 μM and 0.20 ± 0.04nmol min-1 nmol-1 CYP, respectively. Coumarin 7-hydroxylation by CYP2A5 was more than 200 times more efficient than 3,4 epoxidation by CYP2A4.
Bibliographical noteFunding Information:
The authors thank Xinxin Ding for providing CYP2A13. The supported by Grant CA-74913 from the National Cancer Institute.