Thermus thermophilus encodes an archaeal-like fructose-1,6-bisphosphatase: Purification of native and recombinant protein for structural studies

Abstract

Fructose-1,6-bisphosphatase is one of the key enzymes of the gluconeogenic pathway. It catalyses the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. Fructose-1,6-bisphosphatase from the extreme thermophilic bacterium Thermus thermophilus has been purified by crystallisation approach. The final well-shaped crystals have been obtained using vapour diffusion sitting-drops in the presence of PEG 400 as the precipitating agent. The initially obtained native twinned crystals diffracted up to 1.2 Å resolution. Untwinned crystals used for data collection, however, were grown in the presence of thiomersal. They diffract to 1.8 Å resolution and belong to the space groups I422 with cell dimensions (i) a = b = 108.8 Å, c = 336.3 Å showing two molecules in the asymmetric unit, and (ii) a = b = 113.7 Å, c = 151.0 Å with one molecule in the asymmetric unit. The crystal structure has been solved by single anomalous dispersion using a 1.9 Å resolution. For further biochemical and biophysical investigations recombinant fructose-1,6-bisphosphatase has been produced in Escherichia coli. Both native (dissolved crystals) and recombinant material have been characterised by SDS–PAGE, N-terminal sequencing and MALDI-MS.