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

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,...

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Bibliographic Details
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
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.82.17.5617