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

  • Alain Mera
  • Mariana Zuliani Theodoro de Lima
  • Amanda Bernardes
  • Wanius Garcia
  • João Renato Carvalho MunizEmail author
Original Article
  • 94 Downloads

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 codifies 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 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.

Keywords

Bacillus licheniformis β-Fructofuranosidase Sucrose-6-phosphate hydrolase Biochemical properties 

Notes

Acknowledgements

We would like to thank the National Synchrotron Light Laboratory (LNLS, Brazil) and Central Experimental Multiusuário da Universidade Federal do ABC (CEM/UFABC); Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support via Grants # 2017/16291-5 (JRCM); # 2017/17275-3 (WG) São Carlos Institute of Physics/University of Sao Paulo; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support via Grants # 309767/2015-6 (JRCM) and 486546/2013-6 (JRCM); and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for fellowship to AM, MZTL.

Compliance with ethical standards

Conflict of interests

All authors of this work declare that they have no potential conflict of interest and that there is no financial, consultant, institutional or other relationships that might lead to bias or conflicts of interest in this research. Financial grants, infrastructure and fellowships supporting this work are described in the acknowledgements section.

Human and animal rights statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Consent to submit this work has been received explicitly from all co-authors, as well as from the institute and the university where the work has been carried out. All authors contributed to the scientific work and, therefore, share collective responsibility and accountability for the results.

Supplementary material

726_2018_2690_MOESM1_ESM.docx (557 kb)
Supplementary material 1 (DOCX 557 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Sao Carlos Institute of Physics (IFSC)University of Sao Paulo (USP)São CarlosBrazil
  2. 2.Mackenzie Presbyterian UniversityCampinasBrazil
  3. 3.Centro de Ciências Naturais e HumanasUniversidade Federal do ABC (UFABC)Santo AndréBrazil

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