New and easy strategy for cloning, expression, purification, and characterization of the 5S subunit of transcarboxylase from Propionibacterium f. shermanii

Institute of Analytical Chemistry, University of Leipzig, Leipzig, Germany.
Preparative Biochemistry & Biotechnology (Impact Factor: 0.91). 02/2007; 37(1):13-26. DOI: 10.1080/10826060601039394
Source: PubMed


Methylmalonyl CoA-oxalacetate transcarboxylase (EC 2. 1. 3. 1) from Propionibacterium f. shermanii is a biotin dependent enzyme which transfers CO2 from methylmalonyl-CoA (MMCoA) to pyruvate via a carboxylated biotin group to form oxalacetate. It is composed of three subunits, the central cylindrical hexameric 12S subunit, the outer six dimeric 5S subunit, and the twelve 1.3S linkers. We here report the cloning, sequencing, expression, and purification of the 5S subunit. The gene was identified by matching the amino acid sequence with that of deposited in the NCBI database. For cloned 5S subunit sequence shows regions of high homology with that of pyruvate carboxylase and oxaloacetate decarboxylase. The gene encoding the 5S subunit was cloned into the pTXB1 vector. The expressed 5S subunit was purified to apparent homogeneity by a single step process by using Intein mediated protein ligation (IPL) method. The cloned 5S gene encodes a protein of 505 amino acids and of M(r) 55,700.

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Available from: Stefan Berger
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    • "This is presumably due to the fact that Bacteroidetes are more abundant than Negativicutes in the human gut microbiota and also carry several copies of the gene (see below). Transcarboxylases, decarboxylases and carboxylases form part of a biotin-enzyme family (Lombard and Moreira, 2011) and the methylmalonyl-CoA decarboxylase alpha subunit (mmdA) from V. parvula shares similarity with the 12S subunit of methylmalonyl-CoA transcarboxylase of the dairy strain Propionibacterium freudenreichii (Kumar Bhat and Berger, 2007) and with the b-subunit of propionyl-CoA carboxylases. Many strains of Bacteroides carry several genes closely related to MmdA (for example, both B. thetaiotaomicron VPI-5482 and B. fragilis YCH46 have three genes with 58–62% identity to MmdA from V. parvula, Supplementary Table S2). "
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