Molecular cloning and expression of cDNA encoding 3alpha,7alpha,12alpha-trihydroxy-5beta-chole stanoyl-CoA oxidase...

The steroid side chain cleavage in bile acid formation is catalyzed by liver peroxisomal enzymes (Pedersen, J. I. and Gustafsson, J. (1980) FEBS Lett. 121, 345-348; Kase, F., Björkhem, I., and Pedersen, J. I. (1983) J. Lipid Res. 24, 1560-1567). We here describe the cloning and sequencing of a cDNA coding the first of these enzymes, a 3alpha,7alpha,12alpha-trihydroxy-5beta-choles tanoyl-CoA oxidase (THCA-CoA oxidase) from rabbit liver peroxisomes. After tryptic digestion of purified protein in a polyacrylamide gel, five peptides were isolated and sequenced. Using two oligonucleotides deduced from the amino acid sequence data, two overlappping clones were isolated from a rabbit liver cDNA library, which together made up a unique cDNA sequence of 2139 base pairs. It contained an open reading frame of 2046 base pairs encoding a protein of 681 amino acids with a molecular mass of 76,209 daltons. All five peptides could be localized within the sequence. Transfection of COS cells with the coding part of the cDNA resulted in a significant expression of THCA-CoA oxidase activity. We were not able to demonstrate 3alpha, 7alpha-dihydroxy-5beta-cholestanoyl-CoA oxidase activity under the same conditions. The obtained sequence showed 73.6% similarity with a proposed rat THCA-CoA oxidase and 81% similarity with a recently reported human branched chain acyl-CoA oxidase, indicating that these three proteins represent the same enzyme. The similarity with rat palmitoyl-CoA oxidase was 41.8%. The C-terminal tripeptide of the protein was SNL, a previously undescribed variant of the main class of peroxisomal targeting signals. Northern blot analysis revealed that the gene is transcribed in liver and kidney, and the major mRNA fraction had a size of approximately 2.6 kilobase pairs.