Human fatty acid omega-hydroxylase, CYP4A11: determination of complete genomic sequence and characterization of purified recombinant protein.
The gene of the human fatty acid omega-hydroxylase, CYP4A11, has been isolated from a human BAC library, and its complete genomic sequence has been determined. The CYP4A11 gene spanned 12,568 bp and contained 12 exons. The known PPAR recognition elements (PPRE), which were reported to be involved in the induction of CYP4A6 by clofibric acid, were not observed within the 5'-flanking region of the CYP4A11 gene. The recombinant CYP4A11 protein expressed in Escherichia coli using the pCWOri expression vector was purified to an almost electrophoretically homogeneous state with a specific content of 6.4 nmol of P450/mg of protein. This P450 exhibited omega-hydroxylation activity toward laurate, with a turnover number of 14.7 nmol/min/nmol of P450. The apparent K(m) and V(max) values were 56.7 microM and 15.2 nmol/min/nmol of P450, respectively. It also showed omega-hydroxylation activity toward palmitate, with a turnover number of 0.78 nmol/min/nmol of P450. Although several reports from other groups described that CYP4A11 preparations catalyzed omega-hydroxylation of arachidonic acid, our purified recombinant protein exhibited no activity toward arachidonic acid nor prostaglandin A(1).
Kawashima
H
H
Department of Urology, Osaka City University Medical School, Osaka, 545-8585, Japan. hidenori@msic.med.osaka-cu.ac.jp
Naganuma
T
T
Kusunose
E
E
Kono
T
T
Yasumoto
R
R
Sugimura
K
K
Kishimoto
T
T
D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D007848:Laurates; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010168:Palmitates / Q000378:metabolism; D010957:Plasmids; D011454:Prostaglandins A / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D014157:Transcription Factors / Q000235:genetics
D007848:Laurates; D010168:Palmitates; D011454:Prostaglandins A; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D014157:Transcription Factors; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; C100573:prostaglandin A1