Bioinformatics-aided function exploration of GH29 fucosidases from human gut Parabacteroides.

Gut microbes produce α-l-fucosidases critical for utilizing human milk oligosaccharides, mucosal and dietary glycans. Although gut Parabacteroides have garnered attention for their impact on host health and disease, their CAZymes remain poorly studied. CAZome analysis of eleven gut Parabacteroides type strains revealed their capacity to degrade mucin O-glycans. Their abundance of GH29 fucosidases caught our attention, and we predicted the functional profiles of 46 GH29 fucosidases using in silico approaches. Our findings showed diverse linkages specificities and species-specific distributions, with over half of GH29 enzymes functioning as α1,3/4 fucosidases, essential for acting on Lewis antigen epitopes of mucin O-glycans. We further enzymatically validated 4 novel GH29 sequences from poorly characterized groups. PgoldGH29A (cluster37 GH29BERT, GH29:75.1 CUPP) does not act on tested natural substrates. PgoldGH29B (cluster1 GH29BERT, GH29:84.1 CUPP) functions as a strict α1,3/4 fucosidase. PgoldGH29C (cluster14 GH29BERT, GH29:29.1 CUPP) displays unprecedented substrate specificity for α1,2/3/4 disaccharides. PgoldGH29D (cluster4 GH29BERT, GH29:6.2 CUPP) acts on α1,2/3/4/6 linkages similar to enzymes from GH29:6.1 CUPP but prefers disaccharides over trisaccharides. These results suggest that PgoldGH29B and PgoldGH29D can contribute to mucin O-glycan degradation via their α1,3/4 and α1,2 fucosidase activity, respectively, while the natural substrates of PgoldGH29A and PgoldGH29C may be irrelevant to host-glycans. These insights enhance our understanding of the ecological niches inhabited by gut Parabacteroides and may guide similar exploration in other intriguing gut microbial species.

Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome.

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.

Substrate specificity and transglycosylation capacity of α-L-fucosidases across GH29 assessed by bioinformatics-assisted selection of functional diversity.

Glycoside hydrolase family 29 (GH29) encompasses α-L-fucosidases, i.e. enzymes that catalyze the hydrolytic release of fucose from fucosylated glycans, including N- and O-linked glycans on proteins, and these α-L-fucosidases clearly play important roles in biology. GH29 enzymes work via a retaining exo-action mechanism, and some can catalyze transfucosylation. There is no formal subfamily division of GH29 α-L-fucosidases, but they are nonetheless divided into two subfamilies: GH29A having a range of substrate specificities and GH29B having narrower substrate specificity. However, the sequence traits that determine the substrate specificity and transglycosylation ability of GH29 enzymes are not well characterized. Here, we present a new functional map of family GH29 members based on peptide-motif clustering via CUPP (conserved unique peptide patterns) and compare the substrate specificity and transglycosylation activity of 21 representative α-L-fucosidases across the 53 CUPP groups identified. The 21 enzymes exhibited different enzymatic rates on 8 test substrates, CNP-Fuc, 2'FL, 3FL, Lewisa, Lewisx, Fuc-α1,6-GlcNAc, Fuc-α1,3-GlcNAc, and Fuc-α1,4-GlcNAc. Certain CUPP groups clearly harbored a particular type of enzymes, e.g. the majority of the enzymes having activity on Lewisa or Lewisx categorized in the same CUPP clusters. In general, CUPP was useful for resolving GH29 into functional diversity subgroups when considering hydrolytic activity. In contrast, the transglycosylation capacity of GH29 α-L-fucosidases was distributed across a range of CUPP groups. Transglycosylation thus appears to be a common trait among these enzymes and not readily predicted from sequence comparison.

Characterization of five marine family 29 glycoside hydrolases reveals an α-L-fucosidase targeting specifically Fuc(α1,4)GlcNAc.

$\text{L} $ -Fucose is the most widely distributed $\text{L} $-hexose in marine and terrestrial environments and presents a variety of functional roles. $\text{L} $-Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae and is found in human milk oligosaccharides (HMOs) and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α-$\text{L} $-fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in Escherichia coli. All five enzymes (Fp231, Fp239, Fp240, Fp251 and Fp284) hydrolyzed the synthetic substrate CNP-α-$\text{L} $-fucose. Assayed against up to 17 fucose-containing oligosaccharides, Fp239 showed activity against the Lewis Y antigen, 2'- and 3-fucosyllactose, while Fp284 degraded 2'-fucosyllactose and Fuc(α1,6)GlcNAc. Furthermore, Fp231 displayed strict specificity against Fuc(α1,4)GlcNAc, a previously unreported specificity in GH29. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6-6.0 and 25°C, hydrolyzing Fuc(α1,4)GlcNAc with kcat = 1.3 s-1 and Km = 660 µM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored.

Utilization Efficiency of Human Milk Oligosaccharides by Human-Associated Akkermansia Is Strain Dependent.

Akkermansia muciniphila is a mucin-degrading bacterium found in the human gut and is often associated with positive human health. However, despite being detected by as early as 1 month of age, little is known about the role of Akkermansia in the infant gut. Human milk oligosaccharides (HMOs) are abundant components of human milk and are structurally similar to the oligosaccharides that comprise mucin, the preferred growth substrate of human-associated Akkermansia. A limited subset of intestinal bacteria has been shown to grow well on HMOs and mucin. We therefore examined the ability of genomically diverse strains of Akkermansia to grow on HMOs. First, we screened 85 genomes representing the four known Akkermansia phylogroups to examine their metabolic potential to degrade HMOs. Furthermore, we examined the ability of representative isolates to grow on individual HMOs in a mucin background and analyzed the resulting metabolites. All Akkermansia genomes were equipped with an array of glycoside hydrolases associated with HMO deconstruction. Representative strains were all able to grow on HMOs with various efficiencies and growth yields. Strain CSUN-19, belonging to the AmIV phylogroup, grew to the highest level in the presence of fucosylated and sialylated HMOs. This activity may be partially related to the increased copy numbers and/or the enzyme activities of the α-fucosidases, α-sialidases, and ß-galactosidases. This study examines the utilization of individual purified HMOs by Akkermansia strains representing all known phylogroups. Further studies are required to examine how HMO ingestion influences gut microbial ecology in infants harboring different Akkermansia phylogroups. IMPORTANCE Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk and provide several benefits to developing infants, including the recruitment of beneficial bacteria to the human gut. Akkermansia strains are largely considered beneficial bacteria and have been detected in colostrum, breast milk, and young infants. A. muciniphila MucT, belonging to the AmI phylogroup, contributes to the HMO deconstruction capacity of the infant. Here, using phylogenomics, we examined the genomic capacities of four Akkermansia phylogroups to deconstruct HMOs. Indeed, each phylogroup contained differences in their genomic capacities to deconstruct HMOs, and representative strains of each phylogroup were able to grow using HMOs. These Akkermansia-HMO interactions potentially influence gut microbial ecology in early life, a critical time for the development of the gut microbiome and infant health.

Fucoidan-active α-L-fucosidases of the GH29 and GH95 families from a fucoidan degrading cluster of the marine bacterium Wenyingzhuangia fucanilytica.

In present work we provide the bioinformatic and biochemical characterization of six α-L-fucosidases that belong to the 29 and 95 families of glycoside hydrolases (GH) from the fucoidan-degrading locus of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. The fucosidases FucWf1GH29, FucWf2GH29, FucWf3GH29 and FucWf6GH29 are relegated to the subfamily A of the GH29 family. The fucosidase FucWf4GH29 bears a distant resemblance to the GH29 and does not belong to either the GH29A or the GH29B subfamilies. Apparently, FucWf4GH29 is the first representative of a new subfamily within the GH29 family of α-L-fucosidases. For the first time the specificity of fucosidases has been studied using a series of fucoidan-related sulfated oligosaccharides. Studied α-L-fucosidases are able to cleave l-fucose from sulfated fucooligosacchrides after their treatment with exo-sulfatases. All studied α-L-fucosidases are cleaving the α-1→3- and α-1→4-linked terminal l-fucose in sulfated fucooligosaccharides. However, only FucWf3GH29 is able to cleave off an α-1→2-linked l-fucose. The fucosidase FucWf5GH95 of the GH95 family is shown to have higher activity on fucoidans than fucosidases of the GH29 family. Supposedly, the α-l-fucosidase FucWf5GH95 participates in fucoidan debranching. The obtained data indicate different roles of fucosidases of the GH29 and GH95 families in the process of fucoidan degradation by the marine bacteria W. fucanilytica CZ1127T.

Functional exploration of the GH29 fucosidase family.

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.

Purification, expression and characterization of a novel α-l-fucosidase from a marine bacteria Wenyingzhuangia fucanilytica.

α-l-Fucosyl residues are frequently found in oligosaccharides, polysaccharides and glycoconjugates which play fundamental roles in various biological processes. α-l-Fucosidases, glycoside hydrolases for catalyzing the removal of α-l-fucose, can serve as desirable tools in the study and the modification of fucose-containing biomolecules. In this study, an α-l-fucosidase named as Alf1_Wf was purified from a marine bacterium Wenyingzhuangia fucanilytica by using a combination of chromatographic procedures. The sequence of Alf1_Wf was identified via proteomics analysis against the predicted proteome of the bacterium. Recombinant Alf1_Wf with 6×His tag was expressed in E. coli and showed α-l-fucosidase activity. Sequence annotation revealed that Alf1_Wf contained a combination of GH29 catalytic domain and CBM35 accessory domain. Alf1_Wf was confirmed as a member of GH29-A subfamily based on the phylogenetic analysis. Furthermore, biochemical properties and kinetic characteristics of the enzyme were also determined. Substrate specificity determination showed that Alf1_Wf was capable in hydrolyzing α1,4-fucosidic linkage and synthetic substrate pNP-fucose. Besides, Alf1_Wf could act on partially degraded fucoidan. This study successfully purified, identified, cloned, expressed and characterized a novel α-l-fucosidase, and meanwhile revealed a new multidomain structure of glycoside hydrolase. The knowledge gained from this study should facilitate the further research and application of α-l-fucosidases.

1,6-α-L-Fucosidases from Bifidobacterium longum subsp. infantis ATCC 15697 Involved in the Degradation of Core-fucosylated N -Glycan.

Bifidobacterium longum subsp. infantis ATCC 15697 possesses five α-L-fucosidases, which have been previously characterized toward fucosylated human milk oligosaccharides containing α1,2/3/4-linked fucose [Sela et al.: Appl. Environ. Microbiol., 78, 795-803 (2012)]. In this study, two glycoside hydrolase family 29 α-L-fucosidases out of five (Blon_0426 and Blon_0248) were found to be 1,6-α-L-fucosidases acting on core α1,6-fucose on the N-glycan of glycoproteins. These enzymes readily hydrolyzed p-nitrophenyl-α-L-fucoside and Fucα1-6GlcNAc, but hardly hydrolyzed Fucα1-6(GlcNAcß1-4)GlcNAc, suggesting that they de-fucosylate Fucα1-6GlcNAcß1-Asn-peptides/proteins generated by the action of endo-ß- N-acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from Cordyceps militaris. To generate homogenous non-fucosylated N-glycan-containing IgG with high antibody-dependent cellular cytotoxicity (ADCC) activity, the resulting GlcNAc-IgG has a potential to be a good acceptor substrate for the glycosynthase mutant of Endo-M from Mucor hiemalis. Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.

Improved Transglycosylation by a Xyloglucan-Active α-l-Fucosidase from Fusarium graminearum.

Fusarium graminearum produces an α-l-fucosidase, FgFCO1, which so far appears to be the only known fungal GH29 α-l-fucosidase that catalyzes the release of fucose from fucosylated xyloglucan. In our quest to synthesize bioactive glycans by enzymatic catalysis, we observed that FgFCO1 is able to catalyze a transglycosylation reaction involving transfer of fucose from citrus peel xyloglucan to lactose to produce 2'-fucosyllactose, an important human milk oligosaccharide. In addition to achieving maximal yields, control of the regioselectivity is an important issue in exploiting such a transglycosylation ability successfully for glycan synthesis. In the present study, we aimed to improve the transglycosylation efficiency of FgFCO1 through protein engineering by transferring successful mutations from other GH29 α-l-fucosidases. We investigated several such mutation transfers by structural alignment, and report that transfer of the mutation F34I from BiAfcB originating from Bifidobacterium longum subsp. infantis to Y32I in FgFCO1 and mutation of D286, near the catalytic acid/base residue in FgFCO1, especially a D286M mutation, have a positive effect on FgFCO1 transfucosylation regioselectivity. We also found that enzymatic depolymerization of the xyloglucan substrate increases substrate accessibility and in turn transglycosylation (i.e., transfucosylation) efficiency. The data include analysis of the active site amino acids and the active site topology of FgFCO1 and show that transfer of point mutations across GH29 subfamilies is a rational strategy for targeted protein engineering of a xyloglucan-active fungal α-l-fucosidase.

Loop engineering of an α-1,3/4-l-fucosidase for improved synthesis of human milk oligosaccharides.

The α-1,3/4-l-fucosidases (EC 3.2.1.111; GH29) BbAfcB from Bifidobacterium bifidum and CpAfc2 from Clostridium perfringens can catalyse formation of the human milk oligosaccharide (HMO) lacto-N-fucopentaose II (LNFP II) through regioselective transfucosylation of lacto-N-tetraose (LNT) with 3-fucosyllactose (3FL) as donor substrate. The current work exploits structural differences between the two enzymes with the aim of engineering BbAfcB into a more efficient transfucosidase and approaches an understanding of structure-function relations of hydrolytic activity vs. transfucosylation activity in GH29. Replacement of a 23 amino acids long α-helical loop close to the active site of BbAfcB with the corresponding 17-aminoacid α-helical loop of CpAfc2 resulted in almost complete abolishment of the hydrolytic activity on 3FL (6000 times lower hydrolytic activity than WT BbAfcB), while the transfucosylation activity was lowered only one order of magnitude. In turn, the loop engineering resulted in an α-1,3/4-l-fucosidase with transfucosylation activity reaching molar yields of LNFP II of 39 ±â€¯2% on 3FL and negligible product hydrolysis. This was almost 3 times higher than the yield obtained with WT BbAfcB (14 ±â€¯0.3%) and comparable to that obtained with CpAfc2 (50 ±â€¯8%). The obtained transfucosylation activity may expand the options for HMO production: mixtures of 3FL and LNT could be enriched with LNFP II, while mixtures of 3FL and lacto-N-neotetraose (LNnT) could be enriched with LNFP III.

Substrate specificity and transfucosylation activity of GH29 α-l-fucosidases for enzymatic production of human milk oligosaccharides.

Human milk oligosaccharides (HMOs) constitute a unique family of bioactive lactose-based molecules present in human breast milk. HMOs are of major importance for infant health and development but also virtually absent from bovine milk used for infant formula. Among the HMOs, the fucosylated species are the most abundant. Transfucosylation catalysed by retaining α-l-fucosidases is a new route for manufacturing biomimetic HMOs. Seven α-l-fucosidases from glycosyl hydrolase family 29 were expressed, characterized in terms of substrate specificity and thermal stability, and shown to be able to catalyse transfucosylation. The α-l-1,3/4-fucosidase CpAfc2 from Clostridium perfringens efficiently catalysed the formation of the more complex human milk oligosaccharide structure lacto-N-fucopentaose II (LNFP II) using 3-fucosyllactose as fucosyl donor and lacto-N-tetraose as acceptor with a 39% yield. α-l-Fucosidases FgFCO1 from Fusarium graminearum and Mfuc5 from a soil metagenome were able to catalyse transfucosylation of lactose using citrus xyloglucan as fucosyl donor. FgFCO1 catalysed formation of 2'-fucosyllactose, whereas Mfuc5 catalysis mainly produced an unidentified, non-HMO fucosyllactose, reaching molar yields based on the donor substrate of 14% and 18%, respectively.

The structure of a glycoside hydrolase 29 family member from a rumen bacterium reveals unique, dual carbohydrate-binding domains.

Glycoside hydrolase (GH) family 29 consists solely of α-L-fucosidases. These enzymes catalyse the hydrolysis of glycosidic bonds. Here, the structure of GH29_0940, a protein cloned from metagenomic DNA from the rumen of a cow, has been solved, which reveals a multi-domain arrangement that has only recently been identified in bacterial GH29 enzymes. The microbial species that provided the source of this enzyme is unknown. This enzyme contains a second carbohydrate-binding domain at its C-terminal end in addition to the typical N-terminal catalytic domain and carbohydrate-binding domain arrangement of GH29-family proteins. GH29_0940 is a monomer and its overall structure consists of an N-terminal TIM-barrel-like domain, a central ß-sandwich domain and a C-terminal ß-sandwich domain. The TIM-barrel-like catalytic domain exhibits a (ß/α)8/7 arrangement in the core instead of the typical (ß/α)8 topology, with the `missing' α-helix replaced by a long meandering loop that `closes' the barrel structure and suggests a high degree of structural flexibility in the catalytic core. This feature was also noted in all six other structures of GH29 enzymes that have been deposited in the PDB. Based on sequence and structural similarity, the residues Asp162 and Glu220 are proposed to serve as the catalytic nucleophile and the proton donor, respectively. Like other GH29 enzymes, the GH29_0940 structure shows five strictly conserved residues in the catalytic pocket. The structure shows two glycerol molecules in the active site, which have also been observed in other GH29 structures, suggesting that the enzyme catalyses the hydrolysis of small carbohydrates. The two binding domains are classed as family 32 carbohydrate-binding modules (CBM32). These domains have residues involved in ligand binding in the loop regions at the edge of the ß-sandwich. The predicted substrate-binding residues differ between the modules, suggesting that different modules bind to different groups on the substrate(s). Enzymes that possess multiple copies of CBMs are thought to have a complex mechanism of ligand recognition. Defined electron density identifying a long 20-amino-acid hydrophilic loop separating the two CBMs was observed. This suggests that the additional C-terminal domain may have a dynamic range of movement enabled by the loop, allowing a unique mode of action for a GH29 enzyme that has not been identified previously.