Cloning and Expression of the 1.3S Biotin-Containing Subunit of Transcarboxylase

• Published in: Proceedings of the National Academy of Sciences - PNAS 1985-09, Vol.82 (17), p.5617-5621
• Main Authors: Murtif, Vicki L., Bahler, Chris R., Samols, David
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Base Sequence; Biochemistry; Biological and medical sciences; Biotechnology; Biotin - metabolism; Carboxyl and Carbamoyl Transferases; Cloning, Molecular; DNA; Enzymes; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gels; Gene Expression Regulation; Genes; Genetic engineering; Genetic technics; Macromolecular Substances; Methods. Procedures. Technologies; Molecular cloning; Oligonucleotide probes; Oligonucleotides; Plasmids; Propionibacterium - genetics; Proteins; Transferases - genetics; Transferases - immunology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.82.17.5617

We have cloned the gene coding for the 1.3S biotin-containing subunit of transcarboxylase (EC 2.1.3.1) from Propionibacterium shermanii. Transcarboxylase is a well-characterized enzyme composed of 30 polypeptides of three different types: twelve 1.3S biotinyl subunits, six 5S dimeric outer subunits, and one 12S hexameric central subunit. In propionic acid fermentation, the enzyme catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate in two partial reactions. The 1.3S subunit binds the outer and central subunits of the enzyme together, and its biotin serves as carboxyl carrier between subsites on the central and outer subunits where each partial reaction occurs. The cloned gene has been expressed in Escherichia coli, and the 1.3S subunit accumulates to 7% of total cellular protein. The foreign protein is recognized and biotinated by biotin holoenzyme synthetase of E. coli. The identifications of the gene and its product were confirmed by four independent approaches: DNA sequence analysis, immunoprecipitation, incorporation of labeled biotin, and measurement of enzymatic activity in the first partial reaction.