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

摘要

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 extemely thermostable since the optimum temperature seemed to be above 100degC.The optimum pH ofr Tk-Fba was determined to be 5.0 in the absence of citrate,while it shifted to around 7.0 in values were determined to be 0.063 mM and 4.37 mM,respectivley.In addition to citrate,phosphoenolypyruvate 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 decreased in the activity to 49%.Tk-Fba also catlayzed the condensation reaction with a similar activity level (14.9 U/mg) to that for FBP cleavage.However,none of the above compounds seemed tohave 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.