| Full text | |
| Author(s): |
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]
Total Authors: 10
|
| Affiliation: | [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
Total Affiliations: 5
|
| Document type: | Journal article |
| Source: | BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS; v. 1865, n. 12, p. 1758-1769, DEC 2017. |
| Web of Science Citations: | 6 |
| Abstract | |
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) | |
| FAPESP's process: | 14/15403-6 - Aspergillus nidulans as a model to manipulate unfolded protein response-related genes |
| Grantee: | Mariane Paludetti Zubieta |
| Support type: | Scholarships in Brazil - Doctorate |
| FAPESP's process: | 12/20549-4 - Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases |
| Grantee: | André Ricardo de Lima Damasio |
| Support type: | Program for Research on Bioenergy (BIOEN) - Young Investigators Grants |
| FAPESP's process: | 13/08293-7 - CCES - Center for Computational Engineering and Sciences |
| Grantee: | Munir Salomao Skaf |
| Support type: | Research Grants - Research, Innovation and Dissemination Centers - RIDC |
| FAPESP's process: | 14/04105-4 - Structural and functional characterization of new cellulases, focusing in the relation between catalytic domains and CBMs |
| Grantee: | Marcelo Vizoná Liberato |
| Support type: | Scholarships in Brazil - Post-Doctorate |
| FAPESP's process: | 14/23051-2 - Comparative analysis of the heterologous protein secretion mechanisms in Aspergillus nidulans |
| Grantee: | Felipe Calzado |
| Support type: | Scholarships in Brazil - Master |
| FAPESP's process: | 08/58037-9 - Library generation for biomass-conversion enzymes from soil metagenome |
| Grantee: | Fábio Márcio Squina |
| Support type: | Program for Research on Bioenergy (BIOEN) - Young Investigators Grants |
| FAPESP's process: | 13/24988-5 - Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases |
| Grantee: | Marcelo Ventura Rubio |
| Support type: | Scholarships in Brazil - Doctorate (Direct) |