Cloning and Expression of a Pig Liver Taurochenodeoxycholic Acid 6α-Hydroxylase (CYP4A21)

Abstract

A cytochrome P450 expressed in pig liver was cloned by polymerase chain reaction using oligonucleotide primers based on amino acid sequences of the purified taurochenodeoxycholic acid 6␣-hydroxylase. This enzyme catalyzes a 6␣-hydroxylation of chenodeoxycholic acid, and the product hyocholic acid is considered to be a primary bile acid specific for the pig. The cDNA encodes a protein of 504 amino acids. The primary structure of the porcine taurochenodeoxycholic acid 6␣-hydroxylase, designated CYP4A21, shows about 75% identity with known members of the CYP4A subfamily in rabbit and man. Transfection of the cDNA for CYP4A21 into COS cells resulted in the synthesis of an enzyme that was recognized by antibodies raised against the purified pig liver enzyme and catalyzed 6␣-hydroxylation of taurochenodeoxycholic acid. The hitherto known CYP4A enzymes catalyze hydroxylation of fatty acids and prostaglandins and have frequently been referred to as fatty acid hydroxylases. A change in substrate specificity from fatty acids or prostaglandins to a steroid nucleus among CYP4A enzymes is notable. The results of mutagenesis experiments indicate that three amino acid substitutions in a region around position 315 which is highly conserved in all previously known CYP4A and CYP4B enzymes could be involved in the altered catalytic activity of CYP4A21.

Profiling of urinary bile acids in piglets by a combination of enzymatic deconjugation and targeted LC-MRM-MS

“…Hence, the identification of MCA as a dominant urinary BA component (81.94 ± 0.70% of FBAs in urine) in the present study is consistent with the literature (6). CDCA is considered to be a precursor of MCA biosynthesis using 6-hydroxylase (CYP4A21), and it is expected that the ratio between MCA and CDCA in pigs is comparable to that of CA and CDCA in most other mammals (6). In the present study, free diOH BAs, including CDCA, were only detected in trace triple conjugated forms in urine suggests that multiple conjugated forms constitute one of the major pathways for the excretion of excess cholesterol.…”

“…An isomer of CA, 3,6,7-trihydroxy-5-cholanoic acid (MCA), is the major BA constituent of porcine bile and considered to be a species-specific primary BA in the pig. Hence, the identification of MCA as a dominant urinary BA component (81.94 ± 0.70% of FBAs in urine) in the present study is consistent with the literature (6). CDCA is considered to be a precursor of MCA biosynthesis using 6-hydroxylase (CYP4A21), and it is expected that the ratio between MCA and CDCA in pigs is comparable to that of CA and CDCA in most other mammals (6).…”

“…1). MCA, an isomer of CA, was identified as a predominant FBA in female piglet urine, which agrees with the previous report that MCA is a species-specific primary BA in the pig (6). CDCA was detected only in trace amounts in urine before the enzymatic assays, but was found in a significant amount after the enzymatic assays, which supports that CDCA is a precursor of MCA in BA biosynthesis in piglets.…”

“…The primary BAs in most mammals are CA and CDCA, and the CYP8B subfamily is required for introduction of a 12-hydroxyl group to CDCA in CA biosynthesis. However, it is known that the domestic pig does not synthesize CA (6). The enzyme (CYP4A21) catalyzing the 6-hydroxylation in MCA biosynthesis, an atypical member of the CYP4A subfamily, was found in pig (6,30).…”

“…However, it is known that the domestic pig does not synthesize CA (6). The enzyme (CYP4A21) catalyzing the 6-hydroxylation in MCA biosynthesis, an atypical member of the CYP4A subfamily, was found in pig (6,30). An isomer of CA, 3,6,7-trihydroxy-5-cholanoic acid (MCA), is the major BA constituent of porcine bile and considered to be a species-specific primary BA in the pig.…”