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

Abstract

Although the genes that encode the glutamyl-tRNA Gin (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 pTregat-CABBST 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 50degreesC. Although Glu-AdTases from both B. subtilis and B. stearothermophilus were subjected to crystallization, the micro-crystals were only obtained from the B.