Cytochrome P450 Enzymes in Bile Acid Biosynthesis and Fatty Acid Metabolism : Studies on Members of the Porcine CYP4A and CYP8B Subfamilies

• Main Author: Lundell, Kerstin
• Format: Doctoral Thesis
• Language: English
• Published: Uppsala universitet, Institutionen för farmaceutisk biovetenskap 2003
• Subjects: Pharmaceutical biochemistry; cytochrome P450; bile acids; fatty acids; CYP4A; CYP8B; genome; porcine; hyocholic acid; Farmaceutisk biokemi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3419; http://nbn-resolving.de/urn:isbn:91-554-5608-1

The present investigation is devoted to studies on porcine members of the cytochrome P450 4A (CYP4A) and CYP8B1 subfamilies, which are involved in bile acid biosynthesis and fatty acid metabolism. Hyocholic acid is considered to fulfil the requirements for trihydroxy bile acids in the domestic pig (Sus scrofa) in the absence of cholic acid. Hyocholic acid is a 6α-hydroxylated product of chenodeoxycholic acid and the enzyme catalyzing the 6α-hydroxylation was cloned and found to be an atypical member of the CYP4A subfamily. The primary structure of this porcine enzyme, designated CYP4A21, shows about 75% overall sequence identity to members of the CYP4A subfamily expressed in rabbit and man. Divergent amino acids in a “signature sequence” in the active site of all hitherto known CYP4A fatty acid hydroxylases, were found to be important determinants for the 6α-hydroxylase activity of CYP4A21. Two homologous CYP4A fatty acid hydroxylases, designated CYP4A24 and CYP4A25, expressed in pig liver and kidney were cloned. These two cDNAs encode proteins of 504 amino acids similar to CYP4A21. The overall identity between CYP4A24 and CYP4A25 is 97% compared to 94% identity to CYP4A21. Whereas CYP4A21 clearly deviates regarding structural features and catalytic activity it is more difficult to establish whether CYP4A24 and CYP4A25 are distinct enzymes or allelic variants of a single enzyme. Cloning of the CYP4A21 gene showed a conserved organization compared to CYP4A genes in other species. A segment of the CYP4A24 gene was also cloned and comparison with the CYP4A21 gene revealed an extensive sequence identity also within introns as well as within the proximal promoter regions. This indicates that CYP4A21 and CYP4A fatty acid hydroxylases have a common origin and evolved by gene duplication. The CYP4A21 and CYP4A fatty acid hydroxylases, however, show distinct patterns of expression. The key enzyme in cholic acid biosynthesis, CYP8B1, was markedly expressed in fetal pig liver compared to livers from young pigs. The opposite was shown for the expression of CYP4A21. An apparently conserved pig CYP8B1 gene was cloned and was intronless, similar to CYP8B1 genes from other species. The pig gene encoded a protein of 501 amino acids with 81% identity to CYP8B1 expressed in rabbit and man. Unlike other CYP8B1 genes, the pig promoter lacked a TATA-box. This might offer one explanation for the unusual expression pattern, which appears to be restricted to pig fetal life.