Extremely stable indole-3-glycerol-phosphate synthase from hyperthermophilic archaeon Pyrococcus furiosus

摘要

The gene-encoding Indole-3-glycerol phosphate synthase, a key enzyme involved in the cyclization of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate, from Pyrococcus furiosus was cloned and expressed in Escherichia coli. The gene product was produced in the soluble and active form. The recombinant protein, purified to apparent homogeneity, displayed highest activity at 100 degrees C and pH of 5.5. The recombinant enzyme followed Michaelis-Menten kinetics exhibiting apparent V-max and K-m values of 20 +/- 0.5molmin(-1) mg(-1) and 140 +/- 10 mu M, respectively. The activation energy, determined from the linear Arrhenius plot, was 17 +/- 0.5kJmol(-1). A unique property of PfInGPS is its stability against denaturants and temperature. There was no significant change in activity even in the presence of 8M urea or 5M guanidine hydrochloride. Furthermore, recombinant PfInGPS was highly thermostable with a half-life of 200min at 100 degrees C. To the best of our knowledge, this is the most stable indole-3-glycerol phosphate synthase characterized to date.