NADPH- and hydroperoxide-supported 17beta-estradiol hydroxylation catalyzed by a variant form (432L, 453S) of human cytochrome P450 1B1.
Human cytochrome P450 1B1 (CYP1B1) catalyzes the hydroxylation of 17beta-estradiol (E(2)) at C-4, with a lesser activity at C-2. The E(2) 4-hydroxylase activity of human CYP1B1 was first observed in studies of MCF-7 breast cancer cells. Sequencing of polymerase chain reaction products revealed that CYP1B1 expressed in MCF-7 cells was not the previously characterized enzyme but a polymorphic form with leucine substituted for valine at position 432 and serine substituted for asparagine at position 453. To investigate the NADPH- and organic hydroperoxide-supported E(2) hydroxylase activities of the 432L, 453S form of human CYP1B1, the MCF-7 CYP1B1 cDNA was cloned and the enzyme was expressed in Sf9 insect cells. In microsomal assays supplemented with human NADPH:cytochrome P450 oxidoreductase, the expressed 432L, 453S form catalyzed NADPH-supported E(2) hydroxylation with a similar preference for 4-hydroxylation as the 432V, 453N form, with maximal rates of 1.97 and 0.37 nmol (min)(-1)(nmol cytochrome P450)(-1) for 4- and 2-hydroxylation, respectively. Cumeme hydroperoxide efficiently supported E(2) hydroxylation by both the 432V, 453N and 432L, 453S forms at several-fold higher rates than the NADPH-supported activities and with a lesser preference for E(2) 4- versus 2-hydroxylation (2:1). The hydroperoxide-supported activities of both forms were potently inhibited by the CYP1B1 inhibitor, 3,3',4, 4',5,5'-hexachlorobiphenyl. These results indicate that the 432V, 453N and 432L, 453S forms of CYP1B1 have similar catalytic properties for E(2) hydroxylation, and that human CYP1B1 is very efficient in catalyzing the hydroperoxide-dependent formation of catecholestrogens.
Spink
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DC
New York State Department of Health, Wadsworth Center, 12201-0509, Albany, NY, USA. spink@wadsworth.org
Spink
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BC
Zhuo
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Hussain
M M
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Gierthy
J F
JF
Ding
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D019943:Amino Acid Substitution / Q000235:genetics*; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001555:Benzene Derivatives / Q000378:metabolism / Q000494:pharmacology*; D015153:Blotting, Western; D002384:Catalysis / Q000187:drug effects; D003001:Cloning, Molecular; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004958:Estradiol / Q000378:metabolism*; D014644:Genetic Variation / Q000235:genetics*; D015698:Genomic Library; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D014407:Tumor Cells, Cultured
D001555:Benzene Derivatives; D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D004958:Estradiol; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; C007164:cumene hydroperoxide