5ubj

Publication Abstract from PubMed

Carbohydrate-Active Enzymes are key enzymes for biomass-to-bioproducts conversion. alpha-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 alpha-L-arabinofuranosidase (AnAbf62Awt) 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 AnAbf62Awt, and we confirmed that the enzyme is N-glycosylated at asparagine 83 by mass spectrometry analysis. The enzyme was also expressed in Escherichia coli and the studies of circular dichroism showed that the melting temperature and structural profile of AnAbf62Awt and the non-glycosylated enzyme from E. coli (AnAbf62Adeglyc) were highly similar. In addition, the designed glycomutant AnAbf62AN83Q presented similar patterns of secretion and activity to the AnAbf62Awt, indicating that the N-glycan does not influence the properties of this enzyme. The crystallographic structure of AnAbf62Adeglyc was obtained and the 1.7A resolution model showed a five-bladed beta-propeller fold, which is conserved in family GH62. Mutants AnAbf62AY312F and AnAbf62AY312S 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.

Structural and functional characterization of a highly secreted alpha-L-arabinofuranosidase (GH62) from Aspergillus nidulans grown on sugarcane bagasse.,Contesini FJ, Liberato MV, Rubio MV, Calzado F, Zubieta MP, Pachon DMR, Squina FM, Bracht F, Skaf MS, Damasio AR Biochim Biophys Acta. 2017 Sep 7. pii: S1570-9639(17)30209-1. doi:, 10.1016/j.bbapap.2017.09.001. PMID:28890404^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.