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

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 K_m 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.