Functional exploration of the GH29 fucosidase family

Abstract

The deoxy sugar l-fucose is frequently found as a glycan constituent on and outside living cells, and in mammals it is involved in a wide range of biological processes including leukocyte trafficking, histo-blood group antigenicity and antibody effector functions. The manipulation of fucose levels in those biomedically important systems may provide novel insights and therapeutic leads. However, despite the large established sequence diversity of natural fucosidases, so far, very few enzymes have been characterized. We explored the diversity of the α-l-fucosidase-containing CAZY family GH29 by bio-informatic analysis, and by the recombinant production and exploration for fucosidase activity of a subset of 82 protein sequences that represent the family’s large sequence diversity. After establishing that most of the corresponding proteins can be readily expressed in E. coli, more than half of the obtained recombinant proteins (57% of the entire subset) showed activity towards the simple chromogenic fucosylated substrate 4-nitrophenyl α-l-fucopyranoside. Thirty-seven of these active GH29 enzymes (and the GH29 subtaxa that they represent) had not been characterized before. With such a sequence diversity-based collection available, it can easily be used to screen for fucosidase activity towards biomedically relevant fucosylated glycoproteins. As an example, the subset was used to screen GH29 members for activity towards the naturally occurring sialyl-Lewis x-type epitope on glycoproteins, and several such enzymes were identified. Together, the results provide a significant increase in the diversity of characterized GH29 enzymes, and the recombinant enzymes constitute a resource for the further functional exploration of this enzyme family.

Nutritional strategies for mucosal health: the interplay between microbes and mucin glycans

“…The highly diverse gut microbiota expresses different enzymes with similar mucus-degrading capabilities. Available genomes from gut microbiota members indicated that a majority of the organisms have the capability to cleave, and catabolize, at least one of the mucin O-glycan monosaccharides [13,18], and more data are becoming available on the characterization of mucin-degrading CAZymes from gut mucosal microbes [13,[19][20][21][22]. For example, the large and diverse glycoside hydrolase 16 (GH16) family were recently characterized as endoacting enzymes and were described to target the polyLacNAc structures within the oligosaccharide side chains of mucins.…”

Biotechnological Production of 2′-Fucosyllactose: A Prevalent Fucosylated Human Milk Oligosaccharide

“…GH29 is the largest fucosidase family, with more than 5000 entries in the CAZy database, but only 34 fucosidases have been characterized. They act through a retaining mechanism and have a broad substrate specificity . The donor substrate is recognized through the nucleophilic amino acid of the enzyme to form the intermediate fucosyl-enzyme, which is not stable and can be hydrolyzed in the presence of water.…”

Architecture Insight of Bifidobacterial α-L-Fucosidases

“…This database classifies fucosidases into three families (GH29, GH95, and GH151) according to their catalytic structures. GH29 fucosidases act through a retaining mechanism and have a broader substrate specificity, including hydrolysis of Fuc-α1,3/4/6 linkages [15]. Moreover, family GH29 fucosidases have been subclassified into two subfamilies.…”