Characterization of L-Glutamine:D-Fructose-6-phosphate Amidotransferase from an Extreme Thermophile Thermus thermophilus HB8

Abstract Glucosamine-6-phosphate synthase from the extremophile Thermus thermophilus (GlmS th ) was purified to homogeneity from an Escherichia coli overproducer. The homodimeric enzyme exhibits an optimum activity at 70°C with a half-life of 90 min at 80°C. Dissociation experiments in guanidinium chloride and urea are consistent with the absence of catalytic activity of the monomer. Differential scanning microcalorimetry analysis of GlmS th revealed an irreversible denaturation process with a Δ H cal = 257 kcal·mol −1 and T m = 82.6°C. Antigenic cross-reaction with GlmS th was observed with the E. coli enzyme using monoclonal antibodies (mAbs) specific for linear epitopes of the glutamine binding domain. However, no cross-reactivity was observed with an mAb specific for a native conformation of the E. coli enzyme. The inhibition constants of 6-diazo-5-oxo- L -norleucine and methoxyfumaroyl- L -2,3-diaminopropionic acid, potent glutamine site-directed affinity labels of the E. coli enzyme, were reduced by 2 to 3 orders of magnitude when tested on GlmS th , whereas the properties of 2-amino-2-deoxy-glucitol-6P, a potent competitive inhibitor of the fructose-6P site, remained unaffected. These data, combined with its unexpected resistance to limited proteolysis, are consistent with an increase in the structural constraint of the thermophile enzyme vs its mesophilic counterpart.