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

April 9, 2019

Title

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

Author

Alain Mera, Mariana Zuliani Theodoro de Lima, Amanda Bernardes, Wanius Garcia, João Renato Carvalho Muniz

Year

2019

Journal

Amino Acids

Abstract

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 codifes a sucrose-6-phosphate hydrolase, a β-fructofuranosidase (E.C. 3.1.2.26, 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 purifed using afnity 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 confrmed 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 fexibility 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 diferential scanning fuorimetry 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.

Instrument

J-815

Keywords

Circular dichroism, Secondary structure, Chemical stability, Biochemistry