Expression, purification, and crystallization of glutamyl-tRNA(Gln) specific amidotransferase from Bacillus stearothermophilus.
Although the genes that encode the glutamyl-tRNA(Gln) (Glu-tRNA(Gln)) 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 overexpressed with pTrcgatCABBST in Escherichia coli retained transamidation activity on the mischarged glutamic acid on the tRNA(Gln). It also produced correctly-charged Gln-tRNA(Gln) at 37, 42, and 50 degrees 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.
Kwak
Joon Hyeok
JH
Hyonam Kidney Laboratory, Soon Chun Hyang University, Seoul 140-743, Korea.
Shin
Kunyoo
K
Woo
Ji-Su
JS
Kim
Mun Kyung
MK
Kim
Sung Il
SI
Eom
Soo Hyun
SH
Hong
Kwang-Won
KW
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003460:Crystallization; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005798:Genes, Bacterial; D015698:Genomic Library; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019884:Nitrogenous Group Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016366:Open Reading Frames; D009876:Operon; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D019884:Nitrogenous Group Transferases; C047694:glutamyl-tRNA(Gln) amidotransferase