8OZU

Fucosidase crystal structure

Summary for 8OZU

• Entry DOI: 10.2210/pdb8ozu/pdb
• Descriptor: Alpha-L-fucosidase, SULFATE ION (3 entities in total)
• Functional Keywords: fucosidase, sugar binding protein
• Biological source: Lacticaseibacillus casei
• Total number of polymer chains: 8
• Total formula weight: 377111.27

Authors

Gallego del Sol, F.,Marina, A. (deposition date: 2023-05-09, release date: 2024-11-20, Last modification date: 2025-06-04)

Primary citation

Becerra, J.E.,Gallego Del Sol, F.,Rubio-Del-Campo, A.,Rodriguez-Diaz, J.,Monedero, V.,Marina, A.,Yebra, M.J.
Unveiling the structural bases of alpha-L-fucosidase B activity through mutants boosting transfucosylation efficiency.
Int.J.Biol.Macromol., 311:143462-143462, 2025

PubMed Abstract: 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 or K 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.

PubMed: 40286956
DOI: 10.1016/j.ijbiomac.2025.143462

Experimental method

X-RAY DIFFRACTION (2.25 Å)