PRIMARY STRUCTURE OF THE MONOMER OF THE 12S-SUBUNIT OF TRANSCARBOXYLASE AS DEDUCED FROM DNA AND CHARACTERIZATION OF THE PRODUCT EXPRESSED IN ESCHERICHIA-COLI

Transcarboxylase from Propionibacterium shermanii is a complex biotin- containing enzyme composed of 30 polypeptides of three different types : a hexameric central 12S subunit to which 6 outer 5S subunits are att ached through 12 1.3S biotinyl subunits. The enzyme catalyzes a two-st ep reaction in which methylmalonyl coenzyme A and pyruvate serve as su bstrates to form propionyl coenzyme A (propionyl-CoA) and oxalacetate, the 12S subunit specifically catalyzing one of the two reactions. We report here the cloning, sequencing, and expression of the 12S subunit . The gene was identified by matching amino acid sequences derived fro m isolated authentic 12S peptides with the deduced sequence of an open reading frame present in a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3S biotinyl subunit. The cloned 12 S gene encodes a protein of 604 amino acids and of M(r) 65,545. The de duced sequence shows regions of extensive homology with the beta subun it of mammalian propionyl-CoA carboxylase as well as regions of homolo gy with acetyl-CoA carboxylase from several species. Two genomic fragm ents were subcloned into pUC19 in an orientation such that the 12S ope n reading frame could be expressed from the lac promoter of the vector . Crude extracts prepared from these cells contained an immunoreactive band on Western blots (immunoblots) which comigrated with authentic 1 2S. The Escherichia coli-expressed 12S was purified to apparent homoge neity by a three-step procedure and compared with authentic 12S from P . shermanii. Their quaternary structures were identical by electron mi croscopy, and the E. coli 12S preparation was fully active in the reac tions catalyzed by this subunit. We conclude that we have cloned, sequ enced, and expressed the 12S subunit which exists in a hexameric activ e form in E. coli.