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J. Biochem, 2003, Vol. 134, No. 4 567-574
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

Genetic, Enzymatic, and Structural Analyses of Phenylalanyl-tRNA Synthetase from Thermococcus kodakaraensis KOD1

Kentaro Shiraki1, Masao Tsuji2, Yoshiteru Hashimoto3, Kenzo Fujimoto1, Shinsuke Fujiwara4, Masahiro Takagi1 and Tadayuki Imanaka*,5

1 School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292; 2 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871; 3 Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572; 4 Department of Bioscience, School of Science and Engineering, Kwansei-Gakuin University, 2-1 Gakuen Sanda, Hyogo 669-1337; and 5 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510

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 ß-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°C and retained its tertiary structure at 98°C. The estimated isoelectric point (pI) for the {alpha}-subunit is 9.4 and that for the ß-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 ß-subunits.

* To whom correspondence should be addressed. Tel: +81-75-383-2777, Fax: +81-75-383-2778, E-mail: imanaka{at}sbchem.kyoto-u.ac.jp


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