Title
The mechanism by which a distinguishing arabinofuranosidase can cope with internal di-substitutions in arabinoxylans
Author
Camila Ramos dos Santos, Priscila Oliveira de Giuseppe, Flávio Henrique Moreira de Souza, Letícia Maria Zanphorlin, Mariane Noronha Domingues, Renan Augusto Siqueira Pirolla, Rodrigo Vargas Honorato, Celisa Caldana Costa Tonoli, Mariana Abrahão Bueno de Morais, Vanesa Peixoto de Matos Martins, Lucas Miranda Fonseca, Fernanda Büchli, Paulo Sergio Lopes de Oliveira, Fábio Cesar Gozzo, Mário Tyago Murakami
Year
2018
Journal
Biotechnology for Biofuels
Abstract
Arabinoxylan is an abundant polysaccharide in industrially relevant biomasses such as sugarcane, corn stover and grasses. However, the arabinofuranosyl di-substitutions that decorate the xylan backbone are recalcitrant to most known arabinofuranosidases (Abfs). In this work, we identified a novel GH51 Abf (XacAbf51) that forms trimers in solution and can cope efficiently with both mono- and di-substitutions at terminal or internal xylopyranosyl units of arabinoxylan. Using mass spectrometry, the kinetic parameters of the hydrolysis of 33-α-l-arabinofuranosyl-xylotetraose and 23,33-di-α-l-arabinofuranosyl-xylotetraose by XacAbf51 were determined, demonstrating the capacity of this enzyme to cleave arabinofuranosyl linkages of internal mono- and di-substituted xylopyranosyl units. Complementation studies of fungal enzyme cocktails with XacAbf51 revealed an increase of up to 20% in the release of reducing sugars from pretreated sugarcane bagasse, showing the biotechnological potential of a generalist GH51 in biomass saccharification. To elucidate the structural basis for the recognition of internal di-substitutions, the crystal structure of XacAbf51 was determined unveiling the existence of a pocket strategically arranged near to the − 1 subsite that can accommodate a second arabinofuranosyl decoration, a feature not described for any other GH51 Abf structurally characterized so far. In summary, this study reports the first kinetic characterization of internal di-substitution release by a GH51 Abf, provides the structural basis for this activity and reveals a promising candidate for industrial processes involving plant cell wall depolymerization.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Thermal stability, Thermodynamics, Protein folding, Protein denaturation, Biochemistry