Tailor-Made α-Glucans by Engineering the Processivity of α-Glucanotransferases via Tunnel-Cleft Active Center Interconversions.

The function of polysaccharides is intimately associated with their size, which is largely determined by the processivity of transferases responsible for their synthesis. A tunnel active center architecture has been recognized as a key factor that governs processivity of several glycoside hydrolases (GHs), e.g.

Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood.

Matching donor and recipient blood groups based on red blood cell (RBC) surface ABO glycans and antibodies in plasma is crucial to avoid potentially fatal reactions during transfusions. Enzymatic conversion of RBC glycans to the universal group O is an attractive solution to simplify blood logistics and prevent ABO-mismatched transfusions. The gut symbiont Akkermansia muciniphila can degrade mucin O-glycans including ABO epitopes.

Insights into the Structure-Function Relationship of GH70 GtfB α-Glucanotransferases from the Crystal Structure and Molecular Dynamic Simulation of a Newly Characterized N1 GtfB Enzyme.

α-Glucanotransferases of the CAZy family GH70 convert starch-derived donors to industrially important α-glucans. Here, we describe characteristics of a novel GtfB-type 4,6-α-glucanotransferase of high enzyme activity (60.8 U mg) from N1 (LrN1 GtfB), which produces surprisingly large quantities of soluble protein in heterologous expression (173 mg pure protein per L of culture) and synthesizes the reuteran-like α-glucan with (α1 → 6) linkages in linear chains and branch points.

Sialidases and fucosidases of Akkermansia muciniphila are crucial for growth on mucin and nutrient sharing with mucus-associated gut bacteria.

The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-secretion by the host, thereby being a key player in mucus turnover. Mucin glycan utilization requires the removal of protective caps, notably fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A.

Butyrate-producing colonic clostridia: picky glycan utilization specialists.

Butyrate-producing human gut microbiota members are recognized for their strong association with a healthy immune-homeostasis and protection from inflammatory disorders and colorectal cancer. These effects are attributed to butyrate, the terminal electron sink of glycan fermentation by prevalent and abundant colonic Firmicutes from the Lachnospiraceae and Oscillospiraceae families. Remarkably, our insight into the glycan utilization mechanisms and preferences of butyrogenic Firmicutes remains very limited as compared with other gut symbionts, especially from the Bacteroides, Bifidobacterium, and Lactobacillus genera.

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins . Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobra.

Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Galβ1-3GalNAc reactivity on Fetuin.

Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum (EngBF) belongs to the glycoside hydrolase family GH101 and has a strict preference towards the mucin type glycan, Galβ1-3GalNAc, which is O-linked to serine or threonine residues on glycopeptides and -proteins. While other enzymes of the GH101 family exhibit a wider substrate spectrum, no GH101 member has until recently been reported to process the α2-3 sialidated mucin glycan, Neu5Acα2-3Galβ1-3GalNAc. However, work published by others (ACS Chem Biol 2021, 16, 2004-2015) during the preparation of the present manuscript demonstrated that the enzymes from several bacteria are able to hydrolyze this glycan from the fluorophore, methylumbelliferyl.

Diversification of a Fucosyllactose Transporter within the Genus .

Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose [FL]), are associated with the formation of a -rich gut microbial community.

Discovery of fungal oligosaccharide-oxidising flavo-enzymes with previously unknown substrates, redox-activity profiles and interplay with LPMOs.

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes.

Structure and evolution of the bifidobacterial carbohydrate metabolism proteins and enzymes.

Bifidobacteria have attracted significant attention because they provide health-promoting effects in the human gut. In this review, we present a current overview of the three-dimensional structures of bifidobacterial proteins involved in carbohydrate uptake, degradation, and metabolism. As predominant early colonizers of the infant's gut, distinct bifidobacterial species are equipped with a panel of transporters and enzymes specific for human milk oligosaccharides (HMOs).

Activity-Based Protein Profiling of Retaining α-Amylases in Complex Biological Samples.

Amylases are key enzymes in the processing of starch in many kingdoms of life. They are important catalysts in industrial biotechnology where they are applied in, among others, food processing and the production of detergents. In man amylases are the first enzymes in the digestion of starch to glucose and arguably also the preferred target in therapeutic strategies aimed at the treatment of type 2 diabetes patients through down-tuning glucose assimilation.

Loss of AA13 LPMOs impairs degradation of resistant starch and reduces the growth of .

Background: Lytic polysaccharide monooxygenases (LPMOs) are often studied in simple models involving activity measurements of a single LPMO or a blend thereof with hydrolytic enzymes towards an insoluble substrate. However, the contribution of LPMOs to polysaccharide breakdown in complex cocktails of hydrolytic and oxidative enzymes, similar to fungal secretomes, remains elusive. Typically, two starch-specific AA13 LPMOs are encoded by mainly Ascomycota genomes.

Butyrate producing colonic Clostridiales metabolise human milk oligosaccharides and cross feed on mucin via conserved pathways.

The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune- and metabolic disorders is enigmatic. Here, we describe catabolic pathways that support the growth of Roseburia and Eubacterium members on distinct human milk oligosaccharides (HMOs).

An 1,4-α-Glucosyltransferase Defines a New Maltodextrin Catabolism Scheme in Lactobacillus acidophilus.

The maltooligosaccharide (MOS) utilization locus in NCFM, a model for human small-intestine lactobacilli, encodes three glycoside hydrolases (GHs): a putative maltogenic α-amylase of family 13, subfamily 20 (GH13_20), a maltose phosphorylase of GH65 (GH65), and a family 13, subfamily 31, member (GH13_31B), annotated as a 1,6-α-glucosidase. Here, we reveal that GH13_31B is a 1,4-α-glucosyltransferase that disproportionates MOS with a degree of polymerization of ≥2, with a preference for maltotriose. Kinetic analyses of the three GHs encoded by the MOS locus revealed that the substrate preference of GH13_31B toward maltotriose complements the ~40-fold lower of GH13_20 toward this substrate, thereby enhancing the conversion of odd-numbered MOS to maltose.

Molecular insight into a new low-affinity xylan binding module from the xylanolytic gut symbiont Roseburia intestinalis.

Efficient capture of glycans, the prime metabolic resources in the human gut, confers a key competitive advantage for gut microbiota members equipped with extracellular glycoside hydrolases (GHs) to target these substrates. The association of glycans to the bacterial cell surface is typically mediated by carbohydrate binding modules (CBMs). Here, we report the structure of RiCBM86 appended to a GH family 10 xylanase from Roseburia intestinalis.

A novel starch-binding laccase from the wheat pathogen Zymoseptoria tritici highlights the functional diversity of ascomycete laccases.

Background: Laccases are multicopper oxidases, which are assigned into auxiliary activity family 1 (AA1) in the CAZy database. These enzymes, catalyzing the oxidation of phenolic and nonphenolic substrates coupled to reduction of O to HO, are increasingly attractive as eco-friendly oxidation biocatalysts. Basidiomycota laccases are well characterized due to their potential in de-lignification of lignocellulose.

Substrate preference of an ABC importer corresponds to selective growth on β-(1,6)-galactosides in subsp. .

Bifidobacteria are exposed to substantial amounts of dietary β-galactosides. Distinctive preferences for growth on different β-galactosides are observed within members, but the basis of these preferences remains unclear. We previously described the first β-(1,6)/(1,3)-galactosidase from subsp.

The human gut Firmicute Roseburia intestinalis is a primary degrader of dietary β-mannans.

β-Mannans are plant cell wall polysaccharides that are commonly found in human diets. However, a mechanistic understanding into the key populations that degrade this glycan is absent, especially for the dominant Firmicutes phylum. Here, we show that the prominent butyrate-producing Firmicute Roseburia intestinalis expresses two loci conferring metabolism of β-mannans.

H, C and N backbone and side-chain assignment of a carbohydrate binding module from a xylanase from Roseburia intestinalis.

The N-terminal domain (residues 28-165) from the glycoside hydrolase family 10 from Roseburia intestinalis (RiCBMx), has been isotopically labeled and recombinantly expressed in Escherichia coli. Here we report H, C and N NMR chemical shift assignments for this carbohydrate binding module (CBM).

Differential bacterial capture and transport preferences facilitate co-growth on dietary xylan in the human gut.

Metabolism of dietary glycans is pivotal in shaping the human gut microbiota. However, the mechanisms that promote competition for glycans among gut commensals remain unclear. Roseburia intestinalis, an abundant butyrate-producing Firmicute, is a key degrader of the major dietary fibre xylan.

Discovery of α-l-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42.

Enzymes of the glycoside hydrolase family 42 (GH42) are widespread in bacteria of the human gut microbiome and play fundamental roles in the decomposition of both milk and plant oligosaccharides. All GH42 enzymes characterized so far have β-galactosidase activity. Here, we report the existence of a GH42 subfamily that is exclusively specific for α-l-arabinopyranoside and describe the first representative of this subfamily.

Effect of repeat unit structure and molecular mass of lactic acid bacteria hetero-exopolysaccharides on binding to milk proteins.

Interactions of exopolysaccharides and proteins are of great importance in food science, but complicated to analyze and quantify at the molecular level. A surface plasmon resonance procedure was established to characterize binding of seven structure-determined, branched hetero-exopolysaccharides (HePSs) of 0.14-4.