A phosphofructokinase homolog from Pyrobaculum calidifontis displays kinase activity towards pyrimidine nucleosides and ribose 1-phosphate

Iram Aziz, Tahira Bibi, Naeem Rashid, Riku Aono, Haruyuki Atomi, Muhammad Akhtar

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus. To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (k cat /K m ) toward R1P was 11.5 mM -1 · s -1 . ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.

Original languageEnglish
Article numbere00284-18
JournalJournal of Bacteriology
Volume200
Issue number16
DOIs
Publication statusPublished - 2018 Aug 1
Externally publishedYes

Fingerprint

Pyrobaculum
Pyrimidine Nucleosides
Phosphofructokinases
Phosphotransferases
Nucleosides
Sugar Phosphates
Desulfurococcaceae
Aeropyrum
Crenarchaeota
Thermococcus
Pentoses
Cytidine
Proteins
Uridine
Archaea
Hominidae
Enzymes
Guanosine Triphosphate
Cell Extracts
Recombinant Proteins

Keywords

  • Archaea
  • Hyperthermophiles
  • Metabolism
  • Nucleoside
  • Nucleoside kinase
  • Pentose
  • Pentose bisphosphate pathway
  • Phosphofructokinase
  • Pyrobaculum
  • Pyrobaculum calidifontis
  • Ribokinase
  • Ribose 1-phosphate kinase

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

A phosphofructokinase homolog from Pyrobaculum calidifontis displays kinase activity towards pyrimidine nucleosides and ribose 1-phosphate. / Aziz, Iram; Bibi, Tahira; Rashid, Naeem; Aono, Riku; Atomi, Haruyuki; Akhtar, Muhammad.

In: Journal of Bacteriology, Vol. 200, No. 16, e00284-18, 01.08.2018.

Research output: Contribution to journalArticle

Aziz, Iram ; Bibi, Tahira ; Rashid, Naeem ; Aono, Riku ; Atomi, Haruyuki ; Akhtar, Muhammad. / A phosphofructokinase homolog from Pyrobaculum calidifontis displays kinase activity towards pyrimidine nucleosides and ribose 1-phosphate. In: Journal of Bacteriology. 2018 ; Vol. 200, No. 16.
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T1 - A phosphofructokinase homolog from Pyrobaculum calidifontis displays kinase activity towards pyrimidine nucleosides and ribose 1-phosphate

AU - Aziz, Iram

AU - Bibi, Tahira

AU - Rashid, Naeem

AU - Aono, Riku

AU - Atomi, Haruyuki

AU - Akhtar, Muhammad

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N2 - The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus. To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (k cat /K m ) toward R1P was 11.5 mM -1 · s -1 . ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.

AB - The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus. To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (k cat /K m ) toward R1P was 11.5 mM -1 · s -1 . ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.

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KW - Pyrobaculum calidifontis

KW - Ribokinase

KW - Ribose 1-phosphate kinase

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