Human CYP4F12 genetic polymorphism: identification and functional characterization of seven variant allozymes.
ABSTRACT The human cytochrome CYP4F12 has been shown to be metabolically active toward inflammatory mediators and exogenous compounds such as antihistaminic drugs. We recently identified a genetic polymorphism within the promoter region, associated with a decreased level of enzyme expression. In the present study, we report the further identification of single nucleotide polymorphisms in the coding sequence of the CYP4F12 gene. A polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of DNA samples from 53 unrelated French Caucasians, allowed the identification of ten mutations, comprising seven missense mutations, 31C>T (Leu11Phe), 38C>T (Pro13Leu), 47C>T (Met16Thr), 4759G>A (Asp76Asn), 4801G>A (Val90Leu), 8896C>T (Arg188Cys) and 23545G>A (Gly522Ser). Their functional impact toward ebastine hydroxylation was evaluated using heterologous expression in Saccharomyces cerevisiae cells of site-directed mutated cDNA variants. Five out seven variants did not exhibit any significant difference in CYP4F12 catalytic activity, whereas two variants, Val90Ile and Arg188Cys, displayed significant changes in their Michaelis-Menten (Km, Vm) parameters. These data on CYP4F12 genetic polymorphism provide tools for further studies of association with pathological processes involving an inflammatory component and with variations in anti-histaminic drug response.
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ABSTRACT: Omega and subterminal hydroxylations of prostaglandins (PGs), leukotriene B4 (LTB4) and some related eicosanoids are catalyzed by the cytochrome P450 (CYP) enzymes belonging to the CYP4A and CYP4F subfamilies. CYP4A4, which is induced in pregnant rabbits, is the only elucidated PGE ω-hydroxylase within the CYP4A subfamily. CYP4F3 is the most tissue specific and most efficient LTB4 ω-hydroxylase, judging from its restricted localization in human polymorphonuclear leukocytes (PMN) and its very low Km value for LTB4. CYP4F2 is widely distributed in human liver and other tissues, and catalyzes ω-hydroxylation of various lipoxygenase-derived eicosanoids as well as LTB4, with relatively comparable and high Km values. CYP4F3B is very similar to CYP4F2 in its tissue localization and its Km value for LTB4. Human seminal vesicle CYP4F8 is the first elucidated hydroxylase with substrate specificity for PG endoperoxides, whereas ram seminal vesicle CYP4F12 is the only elucidated PGE ω-hydroxylase within the CYP4F subfamily. Rat CYP4F1, CYP4F4 and CYP4F5, and mouse Cyp4f14 have LTB4 ω-hydroxylase activity. Three additional human, four mouse, and one fish members of the CYP4F subfamily have been identified.Prostaglandins & other lipid mediators 01/2003; 70(s 3–4):361. · 2.42 Impact Factor
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ABSTRACT: A cDNA encoding a novel human CYP4F enzyme (designated CYP4F12) was cloned by PCR from a human small intestine cDNA library. RT-PCR analysis demonstrated that CYP4F12 is expressed in human small intestine and liver. This cDNA contains an entire coding region of a 524-amino-acid protein that is 81.7, 78.3, and 78.2% identical to CYP4F2, CYP4F3, and CYP4F8, respectively. When expressed in Saccharomyces cerevisiae, the P450 catalyzes leukotriene B4 ω-hydroxylation and arachidonic acid ω-hydroxylation, typical reactions of CYP4F isoforms. Their activity levels are, however, much lower than those of CYP4F2. Interestingly, CYP4F12 catalyzes the hydroxylation of the antihistamine ebastine with significantly higher catalytic activity relative to CYP4F2 (385 vs 5 pmol/min/nmol P450). These results indicate that CYP4F12 has a different profile of substrate specificity from other CYP4F isoforms, enzymes responsible for metabolizing endogenous autacoids, therefore suggesting that it may play an important role in xenobiotic biotransformation in the human small intestine.Biochemical and Biophysical Research Communications 03/2001; · 2.41 Impact Factor
- Methods in Enzymology 02/1996; 272:51-64. · 2.00 Impact Factor