Expression, purification, and crystallization of glutamyl-tRNA Gln specific amidotransferase from Bacillus stearothermophilus

Joon Hyeok Kwak, Kunyoo Shin, Ji Su Woo, Mun Kyung Kim, Sung Il Kim, Soo Hyun Eom, Hong Kwang-Won

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)374-381
Number of pages8
JournalMolecules and Cells
Volume14
Issue number3
StatePublished - Dec 2002
Externally publishedYes

Fingerprint

RNA, Transfer, Gln
Geobacillus stearothermophilus
Crystallization
Bacillus subtilis
Open Reading Frames
Enzymes
Gene Order
glutamyl-tRNA(Gln) amidotransferase
Operon
Multigene Family
Organelles
Genes
Staphylococcus aureus
Organism Cloning
Glutamic Acid
Adenosine Triphosphate
Escherichia coli
Bacteria

Keywords

  • Bacillus stearothermophilus gatCAB
  • Glu-tRNA Amidotransferase (Glu-AdTase)
  • Purification and Crystallization

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Kwak, J. H., Shin, K., Woo, J. S., Kim, M. K., Kim, S. I., Eom, S. H., & Kwang-Won, H. (2002). Expression, purification, and crystallization of glutamyl-tRNA Gln specific amidotransferase from Bacillus stearothermophilus. Molecules and Cells, 14(3), 374-381.

Expression, purification, and crystallization of glutamyl-tRNA Gln specific amidotransferase from Bacillus stearothermophilus. / Kwak, Joon Hyeok; Shin, Kunyoo; Woo, Ji Su; Kim, Mun Kyung; Kim, Sung Il; Eom, Soo Hyun; Kwang-Won, Hong.

In: Molecules and Cells, Vol. 14, No. 3, 12.2002, p. 374-381.

Research output: Contribution to journalArticle

Kwak, JH, Shin, K, Woo, JS, Kim, MK, Kim, SI, Eom, SH & Kwang-Won, H 2002, 'Expression, purification, and crystallization of glutamyl-tRNA Gln specific amidotransferase from Bacillus stearothermophilus', Molecules and Cells, vol. 14, no. 3, pp. 374-381.
Kwak, Joon Hyeok ; Shin, Kunyoo ; Woo, Ji Su ; Kim, Mun Kyung ; Kim, Sung Il ; Eom, Soo Hyun ; Kwang-Won, Hong. / Expression, purification, and crystallization of glutamyl-tRNA Gln specific amidotransferase from Bacillus stearothermophilus. In: Molecules and Cells. 2002 ; Vol. 14, No. 3. pp. 374-381.
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AB - 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.

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