Abstract
Although the genes that encode the glutamyl-tRNAGln (Glu-tRNAGln) specific amidotransferase (Glu-AdTase) from various bacteria and eukaryotic organelles are known, the precise mechanism of the enzyme is still unclear. One of the reasons is that there is no information on the three-dimensional structure of the complex, the Glu-AdTase:Glu-tRNA Gln:ATP:amino group donor. To obtain the crystals of Glu-AdTase, the Glu-AdTase of Bacillus stearothermophilus was overexpressed and purified after cloning of the gene that encodes the enzyme. The cloned DNA contained the full-length gene cluster that represented the Glu-AdTase of B. stearothermophilus, and was organized as an operon that consisted of three open-reading frames (ORFs). The order of the genes was gatCAB, as shown in Bacillus subtilis. The ORFs showed a high amino-acid homology to those of B. subtilis (A subunit, 73.2%; B subunit, 81.6%; C subunit, 69.5%) and Staphylococcus aureus (A subunit, 61.9%; B subunit, 71.8%; C subunit, 45.9%). The ORFs were re-cloned on the overexpression vector, pTrc99a, and a recombinant pTrcgatCABBST was obtained. The Glu-AdTase that was overexpresscd with pTrcgatCABBST in Escherichia coli retained transamidation activity on the mischarged glutamic acid on the tRNAGln. It also produced correctly-charged Gln-tRNAGln at 37, 42, and 50°C. Although Glu-AdTases from both B. subtilis and B. stearothermophilus were subjected to crystallization, the micro-crystals were only obtained from the B. stearothermophilus enzyme.
Original language | English (US) |
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Pages (from-to) | 374-381 |
Number of pages | 8 |
Journal | Molecules and Cells |
Volume | 14 |
Issue number | 3 |
State | Published - Dec 2002 |
Externally published | Yes |
Keywords
- Bacillus stearothermophilus gatCAB
- Glu-tRNA Amidotransferase (Glu-AdTase)
- Purification and Crystallization
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology