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

Autor(es)

Mera, Alain ^[1] ; Theodoro de Lima, Mariana Zuliani ^{[1, 2]} ; Bernardes, Amanda ^[1] ; Garcia, Wanius ^[3] ; Carvalho Muniz, Joao Renato ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Sao Carlos Inst Phys IFSC, Ave Joao Dagnone, 1100, BR-13563120 Sao Carlos, SP - Brazil

^[2] Univ Prebiteriana Mackenzie, Campinas, SP - Brazil

^[3] Univ Fed ABC, Ctr Ciencias Nat & Humanas, Santo Andre, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Amino Acids; v. 51, n. 4, p. 599-610, APR 2019.
• Citações Web of Science: 0

Resumo

Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like -fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a -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 degrees C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a -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 -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. (AU)

• Processo FAPESP: 17/17275-3 - Estudos do modo de ação de duas mono-oxigenases líticas de polissacarídeos de inseto (ordem isoptera): estrutura molecular, química bioinorgânica e aplicações biotecnológicas
• Beneficiário: Wanius José Garcia da Silva
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 17/16291-5 - Caracterização biofísica-estrutural de enzimas termofílicas prospectadas do fungo Thielavia terrestris para degradação de biomassa e geração de produtos biotecnológicos
• Beneficiário: João Renato Carvalho Muniz
• Modalidade de apoio: Auxílio à Pesquisa - Regular