Gene cloning and characterization of fructose-1,6-bisphosphate aldolase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

Hiroyuki Imanaka, Toshiaki Fukui, Haruyuki Atomi, Tadayuki Imanaka

Research output: Contribution to journalArticle

20 Citations (Scopus)


The fructose-1,6-bisphosphate (FBP) aldolase gene from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned. The gene encoding FBP aldolase (Tk-fba) was expressed in Escherichia coli and the purified recombinant protein was characterized at high temperature. Tk-Fba is a homodecamer with a subunit molecular mass of 31,283 Da. The amino acid sequence, decameric conformation, formation of a Schiff-base intermediate, and stimulation (286%) of FBP cleavage activity by citrate suggested that Tk-Fba belonged to Class IA, a subtype of the classical Class I aldolases. The specific activity for the FBP cleavage reaction was 18.9 U/mg, which was much higher than those of other Class IA type FBP aldolases. Tk-Fba was extremely thermostable since the optimum temperature seemed to be above 100°C. The optimum pH for Tk-Fba was determined to be 5.0 in the absence of citrate, while it shifted to around 7.0 in the presence of citrate. Tk-Fba accepted FBP and fructose-1-phosphate as substrates and Km values were determined to be 0.063 mM and 4.37 mM, respectively. In addition to citrate, phosphoenolpyruvate and pyrophosphate were also found to be potent activators of Tk-Fba, enhancing activities up to 346% and 201%, respectively. Erythrose-4-phosphate acted as an inhibitor and caused a decrease in the activity to 49%. Tk-Fba also catalyzed the condensation reaction with a similar activity level (14.9 U/mg) to that for FBP cleavage. However, none of the above compounds seemed to have a significant effect on the condensation reaction by Tk-Fba. These results suggest a regulatory function of Tk-Fba toward the catabolic direction of sugar metabolism in T. kodakaraensis KOD1.

Original languageEnglish
Pages (from-to)237-243
Number of pages7
JournalJournal of Bioscience and Bioengineering
Issue number3
Publication statusPublished - 2002
Externally publishedYes



  • Archaea
  • Fructose-1,6-bisphosphate aldolase
  • Glycolysis
  • Hyperthermophile
  • Thermococcus kodakaraensis KOD1

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering

Cite this