Molecular cloning and expression of cDNA encoding 3alpha,7alpha,12alpha-trihydroxy-5beta-chole stanoyl-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) 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.
Pedersen
J I
JI
Division of Clinical Chemistry, Karolinska Institute, Huddinge University Hospital, 14186 Huddinge, Sweden. j.i.pedersen@basalmed.uio.no
Eggertsen
G
G
Hellman
U
U
Andersson
U
U
Björkhem
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D018076:DNA, Complementary; D011994:Recombinant Proteins; D010088:Oxidoreductases; C066160:trihydroxycholestanoyl-CoA oxidase