Cloning and characterization of CYP4F21: A prostaglandin E-2 20-hydroxylase of ram seminal vesicles

Ram semen contains high concentrations of PGE(1), PGE(2), 20-hydroxy-PGE(1) , and 20-hydroxy-PGE(2), which mainly originate from the ram seminal vesicl es. The 20-hydroxy-PGE compounds are formed by a tentatively identified cyt ochrome P450, designated PGE(2) 20-hydroxylase. Our aim was to clone the en zyme and express it in yeast. Total RNA was isolated from ram seminal vesic le. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerat e 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 b y liquid chromatography-mass spectrometry. Recombinant CYP4F21 oxidizes thr ee stable PGH(2) analogs (U44069, U46619, and U51605) and PGE(2) to their 2 0-hydroxy metabolites, whereas PGH(1), PGH(2), PGE(1), and PGF(2 alpha) app eared to be poor substrates. The apparent K-m for hydroxylation of PGE(2) w as 0.05 mM. Microsomes of ram seminal vesicles and NADPH metabolized PGE(2) and the three PGH(2) analogs essentially in the same way as CYP4F21. Our r esults suggest that CYP4F21 might be a sheep homolog to CYP4F8 of human sem inal vesicles. The reproductive function of CYP4F21 is likely to biosynthes ize 20-hydroxy-PGE(1) and 20-hydroxy-PGE(2), which is excreted by the semin al vesicles. (C) 2001 Academic Press.