Publications

• Structure and chromosomal assignment of the sterol 12alpha-hydroxylase gene (CYP8B1) in human and mouse: eukaryotic cytochrome P-450 gene devoid of introns.

  Gaafvels M., Olin M., Chowdhary B.P., Raudsepp T., Andersson U., Persson B., Jansson M., Bjoerkhem I., Eggertsen G.

  Sterol 12alpha-hydroxylase (CYP8B1) is a hepatic cytochrome P-450 that controls the ratio of cholic acid over chenodeoxycholic acid in bile and thus controls the solubility of cholesterol. Both the human and the mouse CYP8B1 complementary DNA and gene were cloned and structurally characterized. Su ... >> More

  Sterol 12alpha-hydroxylase (CYP8B1) is a hepatic cytochrome P-450 that controls the ratio of cholic acid over chenodeoxycholic acid in bile and thus controls the solubility of cholesterol. Both the human and the mouse CYP8B1 complementary DNA and gene were cloned and structurally characterized. Surprisingly, the genomic DNA from both species was found to lack introns. The major transcript of the human gene was estimated to be 3950 bp, and the putative promoter region was estimated to be at least 1360 bp. The murine structural gene was found to span approximately 3 kb. By using FISH and radiation hybrid mapping techniques, the human CYP8B1 gene was located to chromosome 3p21.3-p22, whereas FISH mapped the murine counterpart to chromosome 9qF4, a region that is homologous to the third human chromosome. The results from the chromosome mapping and Southern blotting indicated that the gene is present in a single copy. Transcription of the mouse and human CYP8B1 genes was initiated from a position situated 51 and 35 bases, respectively, downstream of a consensus TATA box. A homology of 21% for the promoter regions of mouse and human may indicate differences in transcriptional regulation. Although a potent induction of CYP8B1 mRNA was observed upon starvation of mice, the mechanism behind this effect was not revealed by analysis of the promoter for potential cis-acting elements. In the human promoter, several possible cis-acting regions were identified but none of them could be directly related to bile acid metabolism. After transfection of COS cells with the human coding region, mRNA and enzymatic activity for the 12alpha-hydroxylase were identified. This is the first mammalian cytochrome P-450 gene reported to lack introns. The importance of this structural feature for evolution and gene regulation is discussed. << Less

  Genomics 56:184-196(1999) [PubMed] [EuropePMC]

• Molecular cloning and expression of rabbit sterol 12alpha-hydroxylase.

  Eggertsen G., Olin M., Andersson U., Ishida H., Kubota S., Hellman U., Okuda K., Bjoerkhem I.

  Sterol 12alpha-hydroxylase is an important enzyme in bile acid biosynthesis, responsible for the balance between formation of cholic acid and chenodeoxycholic acid. The enzyme has been purified to apparent homogeneity from rabbit liver (Ishida, H., Noshiro, M., Okuda, K., and Coon, M. J. (1992) J. ... >> More

  Sterol 12alpha-hydroxylase is an important enzyme in bile acid biosynthesis, responsible for the balance between formation of cholic acid and chenodeoxycholic acid. The enzyme has been purified to apparent homogeneity from rabbit liver (Ishida, H., Noshiro, M., Okuda, K., and Coon, M. J. (1992) J. Biol. Chem. 267, 21319-21323), and we here describe the cloning and sequencing of a cDNA coding for this enzyme. After tryptic digestion of purified protein in a polyacrylamide gel, eight different peptides were isolated and sequenced. Using oligonucleotides deduced from the amino acid sequences, clones were isolated from a rabbit liver cDNA library. In addition to several overlapping clones, one full-length clone was obtained that coded for a polypeptide of 500 amino acids, corresponding to a molecular mass of 57 kDa. All of the eight peptides and the reported NH2-terminal amino acid sequence were matched against the sequence. The peptide sequence showed a 39% similarity with human prostacyclin synthase (CYP8) and 31% similarity with the rate-limiting enzyme in over-all synthesis of bile acids, the cholesterol 7alpha-hydroxylase (CYP7) of the rabbit. The similarity with most other sterol cytochrome P-450 hydroxylases was less. Thus, this species of cytochrome P-450 should belong to a group of its own, here denoted CYP12. Transfection of COS cells with the coding part of the cDNA resulted in a significant expression of sterol 12alpha-hydroxylase activity toward 7alpha-hydroxy-4-cholesten-3-one. Northern blotting showed that the enzyme was exclusively expressed in the liver. The major mRNA fraction in rabbit liver had a size of approximately 2.9 kilobases, and those found in rat and human liver were about 2.5 and 4.5 kilobases, respectively. Fasting of rats and mice led to a severalfold increase in both enzyme activity and mRNA levels. In contrast, starvation of rabbits had little or no stimulatory effect on enzyme activity and mRNA levels. << Less

  J. Biol. Chem. 271:32269-32275(1996) [PubMed] [EuropePMC]

• Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase.

  Ishida H., Noshiro M., Okuda K., Coon M.J.

  The isoform of cytochrome P450 that catalyzes the 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one, an intermediate in the conversion of cholesterol to cholic acid, was purified to homogeneity from rabbit liver microsomes. The extent of purification in the various steps was judged by an ... >> More

  The isoform of cytochrome P450 that catalyzes the 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one, an intermediate in the conversion of cholesterol to cholic acid, was purified to homogeneity from rabbit liver microsomes. The extent of purification in the various steps was judged by an assay involving high performance liquid chromatography. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis (M(r) = 50,000). The NH2-terminal amino acid sequence is as follows: Val-Leu-Trp-Gly-Leu-Leu-Gly-Ala-Leu-Leu-Met-Val-Met-Val-Gly-, which is different from that of any other P450s so far reported. The specific content of the enzyme was 13.3 nmol of cytochrome P450/mg of protein. Upon reconstitution with NADPH-cytochrome P450 reductase and cytochrome b5, the P450 enzyme showed a high activity of 12 alpha-hydroxylation with a turnover number of 36.6 min-1 at 37 degrees C. The omission of either cytochrome P450 or NADPH-cytochrome P450 reductase resulted in complete loss of activity, and the omission of cytochrome b5 resulted in 40% loss of activity. Antibodies prepared from mouse inhibited the 12 alpha-hydroxylase activity of rabbit liver microsomes about 90% and that of the rat liver microsomes 50%. The enzyme activity was not inhibited by other antibodies raised against other forms of P450 that catalyze different monooxygenation reactions toward xenobiotics or endogenous substrates. Anti-cytochrome b5 antibody inhibited the activity 40%, suggesting the functional role of this protein, and anti-reductase inhibited the activity almost completely. The microsomal enzyme activity was markedly elevated by starvation or streptozotocin administration to the animals. However, an immunoblotting experiment showed no correlation between the enzyme activity and the amount of protein, suggesting that post-translational modification may occur. << Less

  J. Biol. Chem. 267:21319-21323(1992) [PubMed] [EuropePMC]

• CYP8B1 Catalyzes 12alpha-Hydroxylation of C&lt;sub&gt;27&lt;/sub&gt; Bile Acid: In Vitro Conversion of Dihydroxycoprostanic Acid into Trihydroxycoprostanic Acid.

  Wang Y., Wang Y., Hu Y., Wu Q., Gui L., Zeng W., Chen Q., Yu T., Zhang X., Lan K.

  Sterol 12α-hydroxylase (CYP8B1) is the unique P450 enzyme with sterol 12-oxidation activity, playing an exclusive role in 12<i>α</i>-hydroxylating intermediates along the bile acid (BA) synthesis pathway. Despite the long history of BA metabolism studies, it is unclear whether CYP8B1 catalyzes 12< ... >> More

  Sterol 12α-hydroxylase (CYP8B1) is the unique P450 enzyme with sterol 12-oxidation activity, playing an exclusive role in 12<i>α</i>-hydroxylating intermediates along the bile acid (BA) synthesis pathway. Despite the long history of BA metabolism studies, it is unclear whether CYP8B1 catalyzes 12<i>α</i>-hydroxylation of C₂₇ BAs, the key intermediates shuttling between mitochondria and peroxisomes. This work provides robust in vitro evidence that both microsomal and recombinant CYP8B1 enzymes catalyze the 12<i>α</i>-hydroxylation of dihydroxycoprostanic acid (DHCA) into trihydroxycoprostanic acid (THCA). On the one hand, DHCA 12<i>α</i>-hydroxylation reactivity is conservatively detected in liver microsomes of both human and preclinical animals. The reactivity of human tissue fractions conforms well with the selectivity of CYP8B1 mRNA expression, while the contribution of P450 enzymes other than CYP8B1 is excluded by reaction phenotyping in commercial recombinant enzymes. On the other hand, we prepared functional recombinant human CYP8B1 proteins according to a recently published protocol. Titration of the purified CYP8B1 proteins with either C4 (7<i>α</i>-hydroxy-4-cholesten-3-one) or DHCA yields expected blue shifts of the heme Soret peak (type I binding). The recombinant CYP8B1 proteins efficiently catalyze 12<i>α</i>-hydroxylation of both DHCA and C4, with substrate concentration occupying half of the binding sites of 3.0 and 1.9 <i>μ</i>M and k_cat of 3.2 and 2.6 minutes^-1, respectively. In summary, the confirmed role of CYP8B1 in 12<i>α</i>-hydroxylation of C₂₇ BAs has furnished the forgotten passageway in the BA synthesis pathway. The present finding might have opened a new window to consider the biology of CYP8B1 in glucolipid metabolism and to evaluate CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases. SIGNIFICANCE STATEMENT: The academic community has spent approximately 90 years interpreting the synthesis of bile acids. However, the 12<i>α</i>-hydroxylation of intermediates catalyzed by CYP8B1 is not completely mapped on the classic pathway, particularly for the C₂₇ bile acids, the pivotal intermediates shuttling between mitochondria and peroxisomes. This work discloses the forgotten 12<i>α</i>-hydroxylation pathway from dihydroxycoprostanic acid into trihydroxycoprostanic acid. The present finding may facilitate evaluating CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases. << Less

  Drug Metab Dispos 52:1234-1243(2024) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The enzymes, regulation, and genetics of bile acid synthesis.

  Russell D.W.

  The synthesis and excretion of bile acids comprise the major pathway of cholesterol catabolism in mammals. Synthesis provides a direct means of converting cholesterol, which is both hydrophobic and insoluble, into a water-soluble and readily excreted molecule, the bile acid. The biosynthetic steps ... >> More

  The synthesis and excretion of bile acids comprise the major pathway of cholesterol catabolism in mammals. Synthesis provides a direct means of converting cholesterol, which is both hydrophobic and insoluble, into a water-soluble and readily excreted molecule, the bile acid. The biosynthetic steps that accomplish this transformation also confer detergent properties to the bile acid, which are exploited by the body to facilitate the secretion of cholesterol from the liver. This role in the elimination of cholesterol is counterbalanced by the ability of bile acids to solubilize dietary cholesterol and essential nutrients and to promote their delivery to the liver. The synthesis of a full complement of bile acids requires 17 enzymes. The expression of selected enzymes in the pathway is tightly regulated by nuclear hormone receptors and other transcription factors, which ensure a constant supply of bile acids in an ever changing metabolic environment. Inherited mutations that impair bile acid synthesis cause a spectrum of human disease; this ranges from liver failure in early childhood to progressive neuropathy in adults. << Less

  Annu. Rev. Biochem. 72:137-174(2003) [PubMed] [EuropePMC]

  This publication is cited by 13 other entries.