Unusual enzyme characteristics of aspartyl-tRNA synthetase from hyperthermophilic archaeon Pyrococcus sp. KOD1.
The aspA gene, encoding the aspartyl-tRNA synthetase (AspRS) from the hyperthermophilic archaeon Pyrococcus sp. KOD1, was expressed in Escherichia coli. The KOD1 AspRS, which was purified to homogeneity and was shown to be functional in dimeric form, aminoacylated tRNA from KOD1. The optimum temperature for this activity was 65 degrees C, which was lower than that for the cell growth of KOD1 (85 degrees C). However, it increased to 75 degrees C by the addition of polyamine molecules, such as putrescine, spermine, and spermidine. Analysis of the thermal denaturations of the enzyme and of KOD1-tRNA indicated that neither of them was denatured at temperatures below 70 degrees C. These results suggest polyamine is one of the factors which are required to stabilize the AspRS-tRNA complex in vivo. In order to determine whether the nucleotide triphosphate (NTP) is required for Asp-tRNA synthesis, the aminoacylation was examined in the presence of each of the four NTPs. AspRS most effectively aminoacylated tRNA in the presence of ATP. However, we also found that the enzyme aminoacylated it even in the presence of GTP and UTP as well. Archaeon synthetase may have an interesting system to utilize other NTPs than ATP. The extreme conditions of early life may have given rise to these unique characteristics which then disappeared from developed organisms through evolution.
Fujiwara
S
S
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan.
Lee
S G
SG
Haruki
M
M
Kanaya
S
S
Takagi
M
M
Imanaka
T
T
D001105:Archaea / Q000201:enzymology*; D001226:Aspartate-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D019143:Evolution, Molecular; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D011073:Polyamines / Q000494:pharmacology; D011489:Protein Denaturation; D012329:RNA, Bacterial / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry; D012349:RNA, Transfer, Asp / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature
D011073:Polyamines; D012329:RNA, Bacterial; D012349:RNA, Transfer, Asp; D011994:Recombinant Proteins; D012265:Ribonucleotides; D012343:RNA, Transfer; D001226:Aspartate-tRNA Ligase