Unveiling the structural bases of α-L-fucosidase B activity through mutants boosting transfucosylation efficiency

The AlfB α-L-fucosidase from Lacticaseibacillus paracasei exhibits high specificity on fucosyl-α1,3-N-acetylglucosamine, achieving yields of 30 % in transfucosylation reactions for its synthesis. By random mutagenesis we selected AlfB variants with enhanced transfucosylation activity. Expression of a collection of alfB mutants in E. coli resulted in the isolation of eighteen clones with reduced activity on p-nitrophenyl-α-L-fucopyranoside. The AlfB variants carried diverse amino substitutions, leading to modifications in their enzymatic parameters. In some cases, these changes increased transfucosylation yields, although no direct correlation was observed between k_cat or K_m values and the yields. One particular AlfB mutant (M58) achieved 100 % yield in the synthesis of fucosyl-α1,3-N-acetylglucosamine. This enzyme contained three amino acid substitutions (N196S, V261M and N346K); however, further analysis confirmed that the N346K mutation was sufficient to generate the maximum yield. Elucidation of the tridimensional structure of AlfB and AlfBM58 through X-ray crystallography allowed us to propose a mechanism by which the mutation at position 346, located in a loop close to the active site of an adjacent monomer in the protein tetramer, enhanced transfucosylation over hydrolysis of fucosyl-α1,3-N-acetylglucosamine. This study paves the way for designing novel AlfB variants as tools for the efficient enzymatic synthesis of regio-specific fucosyl-oligosaccharides of biotechnological interest.