Improvement of the versatility of an arabinofuranosidase against galactofuranose for the synthesis of galactofuranoconjugates

Abstract

A new performant biocatalyst was developed for the synthesis of O-, S- and acyl-galactofuranoconjugates.

Modulation of the Activity and Regioselectivity of a Glycosidase: Development of a Convenient Tool for the Synthesis of Specific Disaccharides

“…In order to compare the motions leading to the different regioisomers rather than finding only the most suitable one, the acceptor was initially positioned in optimized ways considering the different (1→X) Man p - p NP approaches instead of a neutral position, and their evolutions over time were evaluated. These in silico studies on the acceptor binding site follow our previous MD investigations about the interactions of the Gal f - p NP donor with the catalytic pocket, in particular with hydrophobic residues [ 9 ]. Considering the very low yield for G(1→2)M disaccharide, its formation in the binding site of enzyme complexes and (1→2) orientation of the acceptor was not considered.…”

“…Ct Ara f 51 is a thermostable arabinofuranosidase from the GH51 family that was shown to be an interesting tool for the synthesis of galactofuranosides [ 7 , 8 , 9 ], with various efficiencies depending on the acceptor substrates. The enzyme follows the typical retaining glycosidase mechanism, i.e., a double-displacement mechanism.…”

“…B-factor colorized maps and the thermal mobilities per residue indicate conformational motions from the α7α7′ loop. Notably, the hydrophobic pocket of the catalytic site with L318 and L319 residues [ 9 ] presented an increased B-factor, in particular with (1→3) and (1→6) orientations ( Supplementary Materials Figure S6A,C ). W296 residue (β7 strand, highly conserved (97%) in GH51 family) enabled conformational modifications by π-stacking and hydrophobic interactions with the acceptor and the other important conserved residue, Y244 (β6 strand) [ 13 ].…”

“…On the basis of our previous computational investigations on the p -nitrophenyl α- l -arabinofuranoside and Gal f - p NP interactions with Ct Ara f 51 subunit [ 9 ], galactofuranosyl-enzyme covalent complex was built on the nucleophilic glutamate E292 with the Yasara Structure module, and optimized by Restricted Hartree–Fock methods [ 18 ]. Geometrical optimizations and energy minimizations were performed by semi-empirical method AM1, coupled with the YAMBER3 force field [ 19 ], an AMBER version for Yasara.…”

“…Due to the complexity of its synthesis, this core heptasaccharide has not yet been evaluated for medical applications. In order to access more easily such kind of galactofuranoconjugates, with a limited number of steps and in a way that follows the concepts of green chemistry, our team developed tools to obtain galactofuranoconjugates through a biocatalytic process involving the use of an arabinofuranosidase from Clostridium thermocellum ( Ct Ara f 51) [ 7 , 8 , 9 ]. In the present study we engineered this enzyme to obtain the disaccharide β- d -galactofuranosyl-(1→3)- d -mannopyranose, the central part of this core heptasaccharide, with a higher regioselectivity and a higher yield.…”