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, 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.
D000595:Amino Acid Sequence; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D019878:Carboxyl and Carbamoyl Transferases; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes; D046911:Macromolecular Substances; D011424:Propionibacterium / Q000235:genetics*; D014166:Transferases / Q000235:genetics* / Q000276:immunology
D046911:Macromolecular Substances; D001710:Biotin; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase