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Low-resolution structure, oligomerization and its role on the enzymatic activity of a sucrose-6-phosphate hydrolase from Bacillus licheniformis

Mera, Alain, de Lima, Mariana Zuliani Theodoro, Bernardes, Amanda, Garcia, Wanius, Muniz, João Renato Carvalho
Amino acids 2019 v.51 no.4 pp. 599-610
Bacillus licheniformis, Escherichia coli, alkalinity, beta-fructofuranosidase, enzyme activity, enzyme stability, fluorometry, genes, light scattering, oligomerization, pH, proteins, small-angle X-ray scattering
Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like β-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a β-fructofuranosidase (E.C., protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0–7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a β-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal β-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.