Coding sequence of the precursor of the β subunit of rat propionyl-CoA carboxylase

Abstract

A cDNA encoding the cytoplasmic precursor of the β subunit of the mitochondrial enzyme propionyl-CoA carboxylase (EC 6.4.1.3) was cloned and sequenced. The DNA sequence of 2070 nucleotides is almost identical in size to the major hybridizing mRNA from rat liver (2000 ± 50 nucleotides), suggesting that the cloned DNA represents nearly all of the mRNA sequence. A polypeptide expressed in vitro from an mRNA transcript of this cDNA is indistinguishable in size from the β subunit precursor (58,500 Da). An open reading frame of 1623 nucleotides, flanked by stop codons, encodes a polypeptide of 541 amino acids; the predicted amino acid sequence was confirmed as that of the β subunit of propionyl-CoA carboxylase by matching it to the amino acid sequences of five peptides derived from pure mature rat enzyme. Although the exact length of the cleavable, NH2-terminal leader peptide has not been determined because the NH2-terminal residue of the mature subunit is blocked, the leader is most likely 40-42 amino acids in length and is highly positively charged. Computer-aided analysis of secondary structure suggests that the leader peptide consists of two α-helical segments, with the two most NH2-terminal arginine residues occupying opposite sites of the first helix; this helix has no apparent hydrophobic moment.