Genetic, enzymatic, and structural analyses of phenylalanyl-tRNA synthetase from Thermococcus kodakaraensis KOD1.
Phenylalanyl-tRNA synthetase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-PheRS) was cloned. The open reading frames for both the alpha-subunit (Tk-pheRSA) and beta-subunit (Tk-pheRSB) genes were 1,503 bp (501 amino acids) and 1,722 bp (574 amino acids), respectively. Tk-pheRSB located 879 bp downstream from Tk-pheRSA with a putative TATA box, suggesting that these two subunits are transcribed and regulated independently in KOD1 cells. Tk-PheRS and its respective subunits were expressed in Escherichia coli cells and the proteins were purified. Tk-PheRS showed an optimum enzymatic activity at around 95 degrees C and retained its tertiary structure at 98 degrees C. The estimated isoelectric point (pI) for the alpha-subunit is 9.4 and that for the beta-subunit is 4.6, the largest difference among the 12 kinds of PheRSs reported. The considerable thermostability of Tk-PheRS may be responsible for the electrostatic interaction between the alpha- and beta-subunits.
Shiraki
Kentaro
K
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292.
Tsuji
Masao
M
Hashimoto
Yoshiteru
Y
Fujimoto
Kenzo
K
Fujiwara
Shinsuke
S
Takagi
Masahiro
M
Imanaka
Tadayuki
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016366:Open Reading Frames; D010652:Phenylalanine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics*; D010802:Phylogeny; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology*; D014158:Transcription, Genetic
D009841:Oligonucleotides; D011994:Recombinant Proteins; D010652:Phenylalanine-tRNA Ligase