Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity.
Activation of 17beta-estradiol (E2) through the formation of catechol estrogen metabolites, 2-OH-E2 and 4-OH-E2, and the C-16alpha hydroxylation product, 16alpha-OH-E2, has been postulated to be a factor in mammary carcinogenesis. Cytochrome P450 1B1 (CYP1B1) exceeds other P450 enzymes in both estrogen hydroxylation activity and expression level in breast tissue. To determine whether inherited variants of CYP1B1 differ from wild-type CYP1B1 in estrogen hydroxylase activity, we expressed recombinant wild-type and five polymorphic variants of CYP1B1: variant 1 (codon 48Arg-->Gly), variant 2 (codon 119Ala-->Ser), variant 3 (codon 432Val-->Leu), variant 4 (codon453Asn-->Ser), variant 5 (48Gly, 119Ser, 432Leu, 453Ser). The His-tagged proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography and analyzed by electrophoresis and spectrophotometry. We performed assays of E2 hydroxylation activity and quantitated production of 2-OH-E2, 4-OH-E2, and 16alpha-OH-E2 by gas chromatography/mass spectrometry. Wild-type CYP1B1 formed 4-OH-E2 as main product (Km, 40+/-8 microM; k(cat) 4.4+/-0.4, min(-1); k(cat)/Km, 110 mM(-1) min(-1)), followed by 2-OH-E2 (Km, 34+/-4 microM; k(cat), 1.9+/-0.1 min(-1); k(cat)/Km, 55 mM(-1)min(-1)) and 16alpha-OH-E2 (Km, 39+/-5.7 microM; k(cat), 0.30+/-0.02 min(-1); k(cat)/Km, 7.6 mM(-1)min(-1)). The CYP1B1 variants also formed 4-OH-E2 as the main product but displayed 2.4- to 3.4-fold higher catalytic efficiencies k(cat)/Km than the wild-type enzyme, ranging from 270 mM(-1)min(-1) for variant 4, to 370 mM(-1)min(-1) for variant 2. The variant enzymes also exceeded wild-type CYP1B1 with respect to 2- and 16alpha-hydroxylation activity. Thus, inherited alterations in CYP1B1 estrogen hydroxylation activity may be associated with significant changes in estrogen metabolism and, thereby, may possibly explain interindividual differences in breast cancer risk associated with estrogen-mediated carcinogenicity.
Hanna
I H
IH
Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Dawling
S
S
Roodi
N
N
Guengerich
F P
FP
Parl
F F
FF
D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D014644:Genetic Variation; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010597:Pharmacogenetics; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D039181:Steroid 16-alpha-Hydroxylase
D003062:Codon; D011994:Recombinant Proteins; D004958:Estradiol; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; D039181:Steroid 16-alpha-Hydroxylase