Cloning and characterization of CYP4F21: a prostaglandin E2 20-hydroxylase of ram seminal vesicles.
Ram semen contains high concentrations of PGE1, PGE2, 20-hydroxy-PGE1, and 20-hydroxy-PGE2, which mainly originate from the ram seminal vesicles. The 20-hydroxy-PGE compounds are formed by a tentatively identified cytochrome P450, designated PGE2 20-hydroxylase. Our aim was to clone the enzyme and express it in yeast. Total RNA was isolated from ram seminal vesicle. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers for the CYP4 family yielded a novel cDNA sequence of a cytochrome P450. The full coding region (1584 bp) was cloned by RT-PCR and designated CYP4F21. The deduced protein sequence of CYP4F21 contained 528 amino acids and showed 74% amino acid identity with CYP4F8 of human seminal vesicles. CYP4F21 was expressed in yeast, and its catalytic properties were studied by liquid chromatography-mass spectrometry. Recombinant CYP4F21 oxidizes three stable PGH2 analogs (U44069, U46619, and U51605) and PGE2 to their 20-hydroxy metabolites, whereas PGH1, PGH2, PGE1, and PGF2alpha appeared to be poor substrates. The apparent Km for hydroxylation of PGE2 was 0.05 mM. Microsomes of ram seminal vesicles and NADPH metabolized PGE2 and the three PGH2 analogs essentially in the same way as CYP4F21. Our results suggest that CYP4F21 might be a sheep homolog to CYP4F8 of human seminal vesicles. The reproductive function of CYP4F21 is likely to biosynthesize 20-hydroxy-PGE1 and 20-hydroxy-PGE2, which is excreted by the seminal vesicles.
Bylund
J
J
Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, Sweden.
Oliw
E H
EH
D000527:Alprostadil / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D015237:Dinoprost / Q000378:metabolism; D015232:Dinoprostone / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism*; D006900:Hydroxylation; D008297:Male; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D044262:Prostaglandin H2; D011463:Prostaglandins H / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012669:Seminal Vesicles / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012756:Sheep
D011463:Prostaglandins H; D011994:Recombinant Proteins; C426764:cytochrome P-450 4F21; D044262:Prostaglandin H2; C048824:20-hydroxyprostaglandin E2; C048825:20-hydroxyprostaglandin E1; D003577:Cytochrome P-450 Enzyme System; D015237:Dinoprost; D006899:Mixed Function Oxygenases; D000527:Alprostadil; D015232:Dinoprostone