MOLECULAR-CLONING AND EXPRESSION OF CDNA-ENCODING -ALPHA,12-ALPHA-TRIHYDROXY-5-BETA-CHOLESTANOYL-COA OXIDASE FROM RABBIT LIVER

The steroid side chain cleavage in bile acid formation is catalyzed by liver peroxisomal enzymes (Pedersen, J. I. and Gustafsson, J. (1980) FEES Lett. 121, 345-348; Kase, F., Bjorkhem, I., and Pedersen, J. I. ( 1983) J. Lipid Res. 24, 1560-1567). We here describe the cloning and s equencing of a cDNA coding the first of these enzymes, a 3 alpha,7 alp ha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA oxidase (THCA-CoA oxida se) from rabbit liver peroxisomes. After tryptic digestion of purified protein in a polyacrylamide gel, five peptides were isolated and sequ enced. Using two oligonucleotides deduced from the amino acid sequence data, two overlapping clones were isolated from a rabbit liver cDNA l ibrary, which together made up a unique cDNA sequence of 2139 base pai rs. It contained an open reading frame of 2846 base pairs encoding a p rotein 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 demonstra te 3 alpha,7 alpha dihydroxy-5 beta-cholestanoyl-CoA oxidase activity under the same conditions. The obtained sequence showed 73.6% similari ty with a proposed rat THCA-CoA oxidase and 81% similarity with a rece ntly reported human branched chain acyl-CoA oxidase, indicating that t hese three proteins represent the same enzyme. The similarity with rat palmitoyl-CoA oxidase was 41.8%. The C-terminal tripeptide of the pro tein was SNL, a previously undescribed variant of the main class of pe roxisomal targeting signals. Northern blot analysis revealed that the gene is transcribed in liver and kidney, and the major mRNA fraction h ad a size of approximately 2.6 kilobase pairs.