Characterization ofL-Glutamine:D-Fructose-6-phosphate Amidotransferase from an Extreme ThermophileThermus thermophilusHB8

Glucosamine-6-phosphate synthase from the extremophileThermus thermophilus(GlmSth) was purified to homogeneity from anEscherichia colioverproducer. 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 GlmSthrevealed an irreversible denaturation process with a ΔHcal= 257 kcal·mol−1andTm= 82.6°C. Antigenic cross-reaction with GlmSthwas observed with theE. colienzyme 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 theE. colienzyme. The inhibition constants of 6-diazo-5-oxo-L-norleucine and methoxyfumaroyl-L-2,3-diaminopropionic acid, potent glutamine site-directed affinity labels of theE. colienzyme, were reduced by 2 to 3 orders of magnitude when tested on GlmSth, 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.