Sterol 27-hydroxylase in bile acid biosynthesis. Mechanism of oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,27-tetrol into 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid.
The sequence of reactions catalyzed by sterol 27-hydroxylase (CYP27) in the oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,27-tetrol into 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid was studied with apparently homogeneous preparations of the cytochrome P-450 from rabbit liver mitochondria. Conditions are described for the formation and characterization of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane-27-al as an enzymatically generated intermediate in the oxidation process. Incubation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol or 5 beta-cholestane-3 alpha,7 alpha,12 alpha,27-tetrol with sterol 27-hydroxylase in 18O2 atmosphere resulted in the incorporation of one or two 18O atoms in the carboxyl group of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid. Similar incubations with 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane-27-al resulted in the incorporation of one 18O atom in the 27-carboxyl group. The results strongly indicate that the sterol 27-hydroxylase performs multiple monooxygenations in the conversion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol into 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid. The following reaction sequence (Reaction 1) at carbon 27 is proposed. [formula: see text] Reaction 1.
Department of Pharmaceutical Biochemistry, University of Uppsala, Sweden.
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I
Holmberg-Betsholtz
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E
Lund
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I
Björkhem
K
K
Wikvall
D000818:Animals; D001647:Bile Acids and Salts / Q000096:biosynthesis*; D053493:Cholestanetriol 26-Monooxygenase; D002777:Cholestanols / Q000138:chemical synthesis / Q000378:metabolism*; D002793:Cholic Acids / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010103:Oxygen Isotopes; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000302:isolation & purification / Q000378:metabolism*; D014316:Tritium
D001647:Bile Acids and Salts; D002777:Cholestanols; D002793:Cholic Acids; D010103:Oxygen Isotopes; D014316:Tritium; C020262:cholestane-3,7,12,26-tetrol; C003681:3,7,12-trihydroxycoprostanic acid; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase