Structural and functional characterization of a highly secreted alpha-L-arabinofuranosidase (GH62) from Aspergillus nidulans grown on sugarcane bagasse

Autor(es)

Contesini, Fabiano Jares ^{[1, 2]} ; Liberato, Marcelo Vizona ^[2] ; Rubio, Marcelo Ventura ^[1] ; Calzado, Felipe ^[1] ; Zubieta, Mariane Paludetti ^[1] ; Riano-Pachon, Diego Mauricio ^{[2, 3]} ; Squina, Fabio Marcio ^[4] ; Bracht, Fabricio ^[5] ; Skaf, Munir S. ^[5] ; Damasio, Andre Ricardo ^[1]

Afiliação do(s) autor(es)

^[1] Univ Estadual Campinas, UNICAMP, Inst Biol, BR-13083862 Campinas, SP - Brazil

^[2] CNPEM, Lab Nacl Ciencia & Tecnol Bioetanol CTBE, Caixa Postal 6192, BR-13083970 Campinas, SP - Brazil

^[3] Univ Sao Paulo, Inst Chem, Dept Biochem, Lab Regulatory Syst Biol, BR-05508000 Sao Paulo, SP - Brazil

^[4] Univ Sorocaba, UNISO, Programa Proc Tecnol & Ambientais, BR-18023000 Sorocaba, SP - Brazil

^[5] Univ Estadual Campinas, UNICAMP, Inst Chem, BR-13084862 Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS; v. 1865, n. 12, p. 1758-1769, DEC 2017.
• Citações Web of Science: 4

Resumo

Carbohydrate-Active Enzymes are key enzymes for biomass-to-bioproducts conversion. a-L-Arabinofuranosidases that belong to the Glycoside Hydrolase family 62 (GH62) have important applications in biofuel production from plant biomass by hydrolyzing arabinoxylans, found in both the primary and secondary cell walls of plants. In this work, we identified a GH62 a-L-arabinofuranosidase (AnAbf62A(wt)) that was highly secreted when Aspergillus nidulans was cultivated on sugarcane bagasse. The gene AN7908 was cloned and transformed in A. nidulans for homologous production of AnAbf62A(wt), and we confirmed that the enzyme is N-glycosylated at asparagine 83 by mass spectrometry analysis. The enzyme was also expressed in Escherichia coil and the studies of circular dichroism showed that the melting temperature and structural profile of AnAbf62A(wt) and the non-glycosylated enzyme from E. coil (AnAbf62A(deglye)) were highly similar. In addition, the designed glycomutant AnAbf62A(N83Q) presented similar patterns of secretion and activity to the AnAbf62A(wt), indicating that the N-glycan does not influence the properties of this enzyme. The crystallographic structure of AnAbf62A(deglyc) was obtained and the 1.7 angstrom resolution model showed a five-bladed beta-propeller fold, which is conserved in family GH62. Mutants AnAbf62A(Y312F) and AnAbf62A(Y312s) showed that Y312 was an important substrate-binding residue. Molecular dynamics simulations indicated that the loop containing Y312 could access different conformations separated by moderately low energy barriers. One of these conformations, comprising a local minimum, is responsible for placing Y312 in the vicinity of the arabinose glycosidic bond, and thus, may be important for catalytic efficiency. (AU)

• Processo FAPESP: 14/23051-2 - Análise comparativa do mecanismo de secreção de proteínas heterólogas em Aspergillus nidulans
• Beneficiário: Felipe Calzado
• Linha de fomento: Bolsas no Brasil - Mestrado
• Processo FAPESP: 12/20549-4 - Secreção de glicoproteínas heterólogas em Aspergillus: efeito do padrão de glicosilação em parâmetros funcionais de glicosil hidrolases
• Beneficiário: André Ricardo de Lima Damasio
• Linha de fomento: Auxílio à Pesquisa - Programa BIOEN - Apoio a Jovens Pesquisadores
• Processo FAPESP: 13/08293-7 - CECC - Centro de Engenharia e Ciências Computacionais
• Beneficiário: Munir Salomao Skaf
• Linha de fomento: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 14/04105-4 - Caracterização estrutural e funcional de novas celulases com foco na relação entre domínios catalíticos e CBMs
• Beneficiário: Marcelo Vizoná Liberato
• Linha de fomento: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 08/58037-9 - Geração de biblioteca para conversão enzimática de biomassa a partir de metagenoma do solo
• Beneficiário: Fábio Márcio Squina
• Linha de fomento: Auxílio à Pesquisa - Programa BIOEN - Apoio a Jovens Pesquisadores
• Processo FAPESP: 14/15403-6 - Aspergillus nidulans como modelo para manipulação de genes envolvidos no processo de "Unfolded Protein Response"
• Beneficiário: Mariane Paludetti Zubieta
• Linha de fomento: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/24988-5 - Secreção de glicoproteínas heterólogas em Aspergillus: efeito do padrão de glicosilação em parâmetros funcionais de glicosil hidrolases
• Beneficiário: Marcelo Ventura Rubio
• Linha de fomento: Bolsas no Brasil - Doutorado Direto