Cloning and expression of a pig liver taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21): a novel member of the CYP4A subfamily.
A cytochrome P450 expressed in pig liver was cloned by polymerase chain reaction using oligonucleotide primers based on amino acid sequences of the purified taurochenodeoxycholic acid 6alpha-hydroxylase. This enzyme catalyzes a 6alpha-hydroxylation of chenodeoxycholic acid, and the product hyocholic acid is considered to be a primary bile acid specific for the pig. The cDNA encodes a protein of 504 amino acids. The primary structure of the porcine taurochenodeoxycholic acid 6alpha-hydroxylase, designated CYP4A21, shows about 75% identity with known members of the CYP4A subfamily in rabbit and man. Transfection of the cDNA for CYP4A21 into COS cells resulted in the synthesis of an enzyme that was recognized by antibodies raised against the purified pig liver enzyme and catalyzed 6alpha-hydroxylation of taurochenodeoxycholic acid. The hitherto known CYP4A enzymes catalyze hydroxylation of fatty acids and prostaglandins and have frequently been referred to as fatty acid hydroxylases. A change in substrate specificity from fatty acids or prostaglandins to a steroid nucleus among CYP4A enzymes is notable. The results of mutagenesis experiments indicate that three amino acid substitutions in a region around position 315 which is highly conserved in all previously known CYP4A and CYP4B enzymes could be involved in the altered catalytic activity of CYP4A21.
Lundell
K
K
Division of Biochemistry, Department of Pharmaceutical Biosciences, University of Uppsala, Sweden. Kerstin.Lundell@farmbio.uu.se
Hansson
R
R
Wikvall
K
K
D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D002384:Catalysis; D002635:Chenodeoxycholic Acid / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013250:Steroid Hydroxylases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D013552:Swine; D014018:Tissue Distribution; D014162:Transfection
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D002635:Chenodeoxycholic Acid; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C095246:taurochenodeoxycholic acid 6-alpha-hydroxylase