title: Enzymatic characterization of in vitro-expressed Baikal seal cytochrome P450 (CYP) 1A1, 1A2, and 1B1: implication of low metabolic potential of CYP1A2 uniquely evolved in aquatic mammals.
abstract: This study aimed to elucidate the catalytic function of cytochrome P450 (CYP) 1 enzymes in aquatic mammals. Alkoxyresorufin O-dealkylation (AROD) activities including methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD), and benzyloxyresorufin O-dealkylation (BROD), and 2- and 4-hydroxylation activities of 17β-estradiol (E2) were measured by using yeast-expressed Baikal seal (Pusa sibirica) CYP1A1, 1A2, and 1B1 proteins. Heterologous protein expression of the Baikal seal CYP1s (bsCYP1s) in yeast microsomes was confirmed by reduced CO-difference spectra and immunoblotting. Heterologously expressed human CYP1 enzyme (hCYP1) activities were simultaneously measured and compared with those of bsCYP1 isozymes. Recombinant bsCYP1A1 protein showed the highest Vmax of EROD, followed by MROD, PROD, and BROD, similar to that of hCYP1A1. Vmax/Km ratios of all AROD activities catalyzed by bsCYP1A1 were lower than those catalyzed by hCYP1A1, suggesting less potential for AROD by bsCYP1A1. Enzymatic assays for bsCYP1A2 showed no or minimal AROD activities, while hCYP1A2 displayed MROD and EROD activities. bsCYP1B1 showed an AROD profile (EROD>BROD>MROD>>PROD) similar to that of hCYP1B1; however, Vmax/Km ratios of all AROD activities by bsCYP1B1 were higher. Yeast microsomes containing bsCYP1A1 and 1B1 and hCYP1A1, 1A2, and 1B1 metabolized E2 to 2-OHE2 and 4-OHE2, whereas bsCYP1A2 showed no such activity. Comparison of 4- and 2-hydroxylations of E2 by CYP1As suggests that bsCYP1A1, hCYP1A1, and 1A2 preferentially catalyze 2- rather than 4-hydroxylation. As for CYP1B1, the Vmax/Km ratios suggest that both Baikal seal and human CYPs catalyze 4- rather than 2-hydroxylation. Interspecies comparison showed that bsCYP1B1 has higher metabolic potencies for both E2 hydroxylations than does hCYP1B1, whereas the activity of bsCYP1A1 was lower than that of hCYP1A1. Messenger RNA expression levels of bsCYP1s in the liver of Baikal seals indicated that bsCYP1A1 and 1A2 enzymes contributed to 16.2% and 83.7% of total CYP1s, respectively; bsCYP1B1 accounted for only 0.06%. Addition of anti-human CYP1A1 antibody in seal liver microsomes suppressed EROD activity more than did anti-human CYP1A2 antibody. Therefore, EROD may be catalyzed by hepatic bsCYP1A1 but not bsCYP1A2, consistent with the results of yeast-expressed bsCYP1A1 and 1A2. In silico substrate-docking models of bsCYP1s suggested that the defect in bsCYP1A2 enzymatic activities may be accounted for by the Pro substitution of highly conserved Thr in the I-helix, which is involved in formation of a hydrogen bond with the hydroperoxy intermediate on the heme. This Thr-Pro substitution is evolutionarily conserved across aquatic mammals and could explain their lower metabolic potential for persistent organic pollutants.
authors: Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan. Electronic address: iwatah@agr.ehime-u.ac.jp.
authors: Hisato
authors: H
authors: Iwata
authors: Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan.
authors: Keisuke
authors: K
authors: Yamaguchi
authors: Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan.
authors: Yoko
authors: Y
authors: Takeshita
authors: Diagnostic Center for Animal Health and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro 080-8555, Japan.
authors: Akira
authors: A
authors: Kubota
authors: Fukuoka Institute of Health and Environmental Sciences, 39 Mukaizano, Dazaifu, Fukuoka 818-0135, Japan.
authors: Shusaku
authors: S
authors: Hirakawa
authors: Center for Environmental Health Sciences, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-8506, Japan.
authors: Tomohiko
authors: T
authors: Isobe
authors: Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan.
authors: Masashi
authors: M
authors: Hirano
authors: Department of Life and Nanopharmaceutical Science and Department of Biology, Kyung Hee University, Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, South Korea. Electronic address: eykim08@khu.ac.kr.
authors: Eun-Young
authors: EY
authors: Kim
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D065633:Cytochrome P-450 CYP1B1 / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism; D005260:Female; D006801:Humans; D006900:Hydroxylation; D008099:Liver / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000378:metabolism; D046048:Seals, Earless / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D014877:Water Pollution, Chemical / Q000009:adverse effects
chemical_list: D015415:Biomarkers; D004967:Estrogens; D013912:Threonine; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D065633:Cytochrome P-450 CYP1B1
keywords: Alkoxyresorufin O-dealkylation; Aquatic mammal; Baikal seal; Conserved threonine; Cytochrome P450; Estrogen hydroxylation