Characterization of L-glutamine:D-fructose-6-phosphate amidotransferase from an extreme thermophile Thermus thermophilus HB8.
Glucosamine-6-phosphate synthase from the extremophile Thermus thermophilus (GlmSth) was purified to homogeneity from an Escherichia coli overproducer. The homodimeric enzyme exhibits an optimum activity at 70 degrees C with a half-life of 90 min at 80 degrees C. Dissociation experiments in guanidinium chloride and urea are consistent with the absence of catalytic activity of the monomer. Differential scanning microcalorimetry analysis of GlmSth revealed an irreversible denaturation process with a delta(H)cal = 257 kcal x mol(-1) and Tm = 82.6 degrees C. Antigenic cross-reaction with GlmSth 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 GlmSth, whereas the properties of 2-amino-2-deoxyglucitol-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.
Badet-Denisot
M A
MA
ICSN-CNRS, UPR 2301, Gif-sur-Yvette, France.
Fernandez-Herrero
L A
LA
Berenguer
J
J
Ooi
T
T
Badet
B
B
D002384:Catalysis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry*; D006358:Hot Temperature; D008970:Molecular Weight; D010449:Peptide Mapping; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*
D011994:Recombinant Proteins; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)