Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus

Akio Kanai, Asako Sato, Yoko Fukuda, Kiyoshi Okada, Takashi Matsuda, Taiichi Sakamoto, Yutaka Muto, Shigeyuki Yokoyama, Gota Kawai, Masaru Tomita

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Using an expression protein library of a hyperthermophilic archaeon, Pyrococcus furiosus, we identified a gene (PF0027) that encodes a protein with heat-stable cyclic nucleotide phosphodiesterase (CPDase) activity. The PF0027 gene encoded a 21-kDa protein and an amino acid sequence that showed ∼27% identity to that of the 2′-5′ tRNA ligase protein, ligT (20 kDa), from Escherichia coli. We found that the purified PF0027 protein possessed GTP-dependent RNA ligase activity and that synthetic tRNA halves bearing 2′,3′-cyclic phosphate and 5′-OH termini were substrates for the ligation reaction in vitro. GTP hydrolysis was not required for the reaction, and GTPγS enhanced the tRNA ligation activity of PF0027 protein, suggesting that the ligation step is regulated by a novel mechanism. In comparison to the strong CPDase activity of the PF0027 protein, the RNA ligase activity itself was quite weak, and the ligation product was unstable during in vitro reaction. Finally, we used NMR to determine the solution structure of the PF0027 protein and discuss the implications of our results in understanding the role of the PF0027 protein.

Original languageEnglish
Pages (from-to)420-431
Number of pages12
JournalRNA
Volume15
Issue number3
DOIs
Publication statusPublished - 2009 Mar

Fingerprint

Pyrococcus furiosus
Archaea
Ligases
Guanosine Triphosphate
Hot Temperature
RNA
Enzymes
Proteins
Ligation
Cyclic Nucleotides
Phosphoric Diester Hydrolases
Transfer RNA
RNA Ligase (ATP)
Genes
Amino Acid Sequence
Hydrolysis
Phosphates
Escherichia coli

Keywords

  • Archaea
  • Cyclic-nucleotide-phosphodiesterase
  • Expression cloning
  • Rna ligase
  • Solution structure

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus. / Kanai, Akio; Sato, Asako; Fukuda, Yoko; Okada, Kiyoshi; Matsuda, Takashi; Sakamoto, Taiichi; Muto, Yutaka; Yokoyama, Shigeyuki; Kawai, Gota; Tomita, Masaru.

In: RNA, Vol. 15, No. 3, 03.2009, p. 420-431.

Research output: Contribution to journalArticle

Kanai, Akio ; Sato, Asako ; Fukuda, Yoko ; Okada, Kiyoshi ; Matsuda, Takashi ; Sakamoto, Taiichi ; Muto, Yutaka ; Yokoyama, Shigeyuki ; Kawai, Gota ; Tomita, Masaru. / Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus. In: RNA. 2009 ; Vol. 15, No. 3. pp. 420-431.
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