Enzymes in different pathways of bile acid biosynthesis

Abstract: Enzymes catalyzing side chain hydroxylations and dehydrogenation/isomerization in different pathways of bile acid biosynthesis were studied.Pig liver mitochondrial sterol 27-hydroxylase (CYP27A) was found to catalyze 24- and 25-hydroxylation of cholesterol in addition to the known 27-hydroxylation. As judged by isotope effect determinations (kH/kD), the 24-hydroxylation is mechanistically different from the 25- and 27-hydroxylations.Biochemical characterization of a cytochrome P450 (51 000-Mr P450) that is separated from CYP27A during purification indicated that this enzyme is a proteolytic degradation product of CYP27A. The 51 000-Mr P450 appears to be formed by endogenous proteases during preparation of mitochondria from some tissues. The isolated cytochrome P450 performed the same reactions as CYP27A but with altered kinetics for some reactions.CYP3A4, a major drug-metabolizing enzyme in human liver, was identified as the major enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol. The 25-hydroxylation is involved in the 25-hydroxylase pathway for cholic acid biosynthesis.A 3β-hydroxy-δ5-C27-steroid dehydrogenase was purified from pig liver microsomes. The enzyme catalyzed the conversion of 7α-hydroxycholesterol, 7α,27-dihydroxycholesterol and 3β,7α-dihydroxy-5-cholestenoic acid into the corresponding 3-oxo-δ4 compounds.The purified 3β-hydroxy-δ5-C27-steroid dehydrogenase was similar to the 3β-hydroxy-D5-steroid dehydrogenases active in steroid hormone biosynthesis regarding N-terminal amino acid sequence. The 3β-hydroxy-δ5-C27-steroid dehydrogenase was not active towards C19- and C21-steroids and the C19/C21-dehydrogenases were not active towards C27-steroids. The 3β-hydroxy-δ5-C27-steroid dehydrogenase was found to be present in liver, lung and ovary microsomes. Immunohistochemical studies showed localization of the enzyme in the bile duct epithelium in the periportal regions.