Cytochrome P450 enzymes in oxygenation of prostaglandin endoperoxides and arachidonic acid : Cloning, expression and catalytic properties of CYP4F8 and CYP4F21

• Cytochrome P450 (P450 or CYP) is an enzyme system involved in the oxygenation of a wide range of endogenous compounds as well as foreign chemicals and drugs. This thesis describes investigations of P450-catalyzed oxygenation of prostaglandins, linoleic and arachidoniacids.The formation of bisallylic hydroxy metabolites of linoleic and arachidonic acids was studied with human recombinant P450s and with human liver microsomes. Several P450 enzymes catalyzed the formation of bisallylic hydroxy metabolites. Inhibition studies and stereochemic analysis of metabolites suggest that the enzyme CYP1A2 may contribute to the biosynthesis of bisallylic hydroxy fatty acid metabolites in adult human liver microsomes.19R-Hydroxy-PGE and 20-hydroxy-PGE are major components of human and ovine semen, respectively. They are formed in the seminal vesicles, but the mechanism of their biosynthesis is unknown. Reverse transcription-polymerase chain reaction using degenerate primers for mammalian CYP4 family genes, revealed expression of two nov P450 genes in human and ovine seminal vesicles. The full coding regions of the genes were cloned and the enzymes were expressed in a yeast system. The human enzyme was designated CYP4F8 and the ovine enzyme was designated CYP4F21. Comparison of their deduced protein sequenceshowed that they had 74 % amino acid identity. Recombinant CYP4F8 oxygenated two prostaglandin endoperoxides (PGH1 and PGH2) and three stable PGH2 analogues int19-hydroxy metabolites. Oxygenation of these substrates was mirrored when incubated with microsomes isolated from human seminal vesicles. These results suggest that CYP4F8 is present in human seminal vesicles and that 19R-hydroxy-PGE is formed by CYP4F8-catalyze 19R-hydroxylation of PGH1 and PGH2, followed by PGE synthase-catalyzed isomerization. Studies of catalytic properties of recombinant CYP4F21 suggest that 20-hydroxy-PGE may be formed by similar mechanisms in ovine seminal vesicles. CYP4F8 is the first enzyme shown to hydroxylate prostaglandin endoperoxides.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Farmaceutiska vetenskaper (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Pharmaceutical Sciences (hsv//eng)

Nyckelord

Pharmaceutical biosciences

Farmaceutisk biovetenskap

Biopharmacy

Biofarmaci

farmaceutisk biokemi

Pharmaceutical Biochemistry

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