Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme

TLDR

Cholesterol is converted to bile acids in the liver via a complex pathway involving at least ten enzymes, with the mitochondrial sterol 26‑hydroxylase catalyzing the first side‑chain oxidation step that initiates bile acid biosynthesis. The study aimed to isolate and characterize a rabbit mitochondrial sterol 26‑hydroxylase cDNA to gain insight into the regulation of bile acid biosynthesis. Protein sequencing, molecular cloning, and expression of the cDNA in monkey COS cells using a versatile eukaryotic vector confirmed the identity of the 26‑hydroxylase. Sequence analysis shows the enzyme is a mitochondrial cytochrome P‑450 with a 36‑residue signal peptide and 499‑amino‑acid mature protein of 56,657 Da, and blotting indicates its mRNA is expressed in many tissues from a low‑copy‑number gene.

Abstract

The conversion of cholesterol into bile acids in the liver represents the major catabolic pathway for the removal of cholesterol from the body. In this complex biosynthetic pathway, at least 10 enzymes modify both the ring structure and side chain of cholesterol, resulting in the formation of the primary bile acids, cholic acid, and chenodeoxycholic acid. To gain insight into the details and regulation of this pathway, we have used protein sequencing and molecular cloning techniques to isolate and characterize a cDNA encoding the rabbit mitochondrial sterol 26-hydroxylase. This enzyme catalyzes the first step in the oxidation of the side chain of sterol intermediates in the biosynthesis of bile acids. The structure of the sterol 26-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, reveals it to be a mitochondrial cytochrome P-450. A signal sequence of 36 residues precedes a coding region of 499 amino acids, predicting a molecular weight of 56,657 for the mature protein. The identity of the 26-hydroxylase cDNA was further confirmed by expression in monkey COS cells employing a versatile eukaryotic expression vector. Blotting experiments revealed that the mRNA for this enzyme is expressed in many tissues and that it is encoded by a low copy number gene in the rabbit genome.