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

Abstract Carbohydrate-Active Enzymes are key enzymes for biomass-to-bioproducts conversion. α- 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 α- l -arabinofuranosidase ( An Abf62A 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 An Abf62A wt , 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 An Abf62A wt and the non-glycosylated enzyme from E . coli ( An Abf62A deglyc ) were highly similar. In addition, the designed glycomutant An Abf62A N83Q presented similar patterns of secretion and activity to the An Abf62A wt , indicating that the N-glycan does not influence the properties of this enzyme. The crystallographic structure of An Abf62A deglyc was obtained and the 1.7 A resolution model showed a five-bladed β-propeller fold, which is conserved in family GH62. Mutants An Abf62A Y312F and An Abf62A 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.