Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence

• Published in: FEBS letters 1993-09, Vol.330 (2), p.191-196
• Main Authors: Thornton, Charles G., Kumar, Ganesh K., Shenoy, Bhami C., Haase, F.Carl, Phillips, Nelson F.B., Park, Vicki M., Magner, William J., Hejlik, Daniel P., Wood, Harland G., Samols, David
• Format: Article
• Language: English
• Subjects: 5 S subunit; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Bacteriology; Base Sequence; Bct, biocytin; Biological and medical sciences; Biotin; Carboxyl and Carbamoyl Transferases; Cloning, Molecular; DNA, Bacterial; Enzymes and enzyme inhibitors; Escherichia coli; Fundamental and applied biological sciences. Psychology; Metabolism. Enzymes; Microbiology; MMCoA, methylmalonyl-CoA; Molecular Sequence Data; Oxalacetate; Propionibacterium - enzymology; Propionibacterium shermanii; Pyruvate; Sequence; Sequence Homology, Amino Acid; TC transcarboxylase; Transcarboxylase; Transferases; Transferases - genetics; WT wild type
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(93)80271-U

Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types. It is composed of six dimeric outer subunits associated with a central cylindrical hexameric subunit through 12 biotinyl subunits; three outer subunits on each face of the central hexamer. Each outer dimer is termed a 5 S subunit which associates with two biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl-CoA and pyruvate form propionyl-CoA and oxalacetate, the 5 S subunit specifically catalyzing one of these reactions. We report here the cloning, sequencing and expression of the monomer of the 5 S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 5 S peptides with the deduced sequence of an open reading frame present on a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3 S biotinyl subunit. The cloned 5 S gene encodes a protein of 519 amino acids, M r, 57,793. The deduced sequence shows regions of extensive homology with that of pyruvate carboxylase and oxalacetate decarboxylase, two enzymes which catalyze the same or reverse reaction. A fragment was subcloned into pUC19 in an orientation such that the 5 S open 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 which co-migrated with authentic 5 S and were fully active in catalyzing the 5 S partial reaction. We conclude that we have cloned, sequenced and expressed the monomer of the 5 S subunit and that the expressed product is catalytically active.