Abstract Polysaccharide utilization loci (PULs) have been a goldmine for the characterization of novel carbohydrate active enzymes (CAZymes) and the understanding of their synergistic degradation of complex polysaccharides. We collected PUL predictions containing CAZymes from glycoside hydrolase families GH29, GH50 and GH117, expected to participate in marine polysaccharide breakdown. We explored the evolutionary diversity in these families in terms of sequences and PUL composition, based on sulfatases and CAZymes. From 41 selected PULs, more than 400 putative enzymes were produced, purified and screened on a large collection of carbohydrates. We attributed a function to more than 130 enzymes from five sulfatase subfamilies, 29 known CAZymes families and discovered an activity for 4 families previously of unknown function, including an α-L-galactosidase structurally and functionally characterized with mutants. Finally, our detailed analysis of the enzymatic synergies in five PULs, two targeting marine polysaccharides and three targeting eukaryotic polysaccharides, by marine and human gut organisms, highlight the efficiency of our exploratory strategy.

Acidic glycans are essential for the biology of multicellular eukaryotes. To utilize them, microbial life including symbionts and pathogens has evolved polysaccharide lyases (PL) that cleave their 1,4 glycosidic linkages via a β-elimination mechanism. PL family 33 (PL33) enzymes have the unusual ability to target a diverse range of glycosaminoglycans (GAGs), as well as the bacterial polymer, gellan gum. In order to gain more detailed insight into PL33 activities we recombinantly expressed 10 PL33 members derived from all major environments and further elucidated the detailed biochemical and biophysical properties of five, showing that their substrate specificity is conferred by variations in tunnel length and topography. The key amino acids involved in catalysis and substrate interactions were identified, and employing a combination of complementary biochemical, structural, and modeling approaches, we show that the tunnel topography is induced by substrate binding to the glycan. Structural and bioinformatic analyses revealed that these features are conserved across several lyase families as well as in mammalian GAG epimerases.

The human gut microbiota can acquire new catabolic functions by integrating genetic material coming from the environment, for example from food-associated bacteria. An illustrative example of that is the acquisition by the human gut microbiota of Asian populations of genes coming from marine bacteria living on the surface of red algae that are incorporated into their diet when eating maki-sushi. To better understand the function and evolution of this set of algal genes corresponding to a polysaccharide utilization locus (PUL) dedicated to the degradation of porphyran, the main polysaccharide of the red algae Porphyra sp., we characterized it biochemically, assessed its genetic diversity and investigated its geographical distribution in large public worldwide datasets. We first demonstrated that both methylated and unmethylated fractions are catabolized without the help of external enzymes. By scanning the genomic data of more than 10,000 cultivated isolates as well as metagenomic data from more than 14,000 worldwide individuals, we found that the porphyran PUL is present in 17 different Phocaeicola/Bacteroides species (including 12 species that were not known to carry it), as well as in two Parabacteroides species and two genera from the Bacillota phylum, highlighting multiple lateral transfers within the gut microbiota. We then analyzed the prevalence of this porphyran PUL across 32 countries and showed that it exists in appreciable frequencies (>1%) only in East Asia (Japan, China, Korea). Finally, we identified three major PUL haplotypes which frequencies significantly differ between these East Asian countries. This geographic structure likely reflects the rate of bacterial horizontal transmission between individuals.

Glycoside Hydrolase family 2 (GH2) is one of the largest and most functionally diverse carbohydrate-active enzyme families. This functional diversity is an obstacle to accurate functional prediction by family assignment and has led to the accumulation of erroneous annotations in non-curated databases. We explored the sequence space of the GH2 family using Sequence-Similarity Networks coupled with closeness centrality to identify 23 subfamilies. The analysis suggests that the GH2 family evolved via multiple duplications followed by neofunctionalization events, with two main activities, β-glucuronidase and β-galacturonidase, re-emerging from likely flexible/reversible ancestors, while an early diverging branch gave birth to several subfamilies with unique activities. To increase the predictive power of subfamily assignments, we biochemically characterized seven members of four of the five subfamilies without previously reported activity. The GH2 subfamilies showing high functional homogeneity will enable more precise functional predictions, while our work highlights subfamilies that require further biochemical and structural investigations.

The salt sensitivity and selectivity feature of α-carrageenan (α-Car) were investigated and compared with κ-carrageenan (κ-Car) and iota-carrageenan (ι-Car). These carrageenans are identified by one sulfate group on the 3,6-anhydro-D-galactose (DA) for α-Car, D-galactose (G) for κ-Car and on both carrabiose moieties (G and DA) for ι-Car. The viscosity and temperature, where order-disorder transition have been observed, were greater in presence of CaCl2 for α-Car and ι-Car compared with KCl and NaCl. Conversely, the reactivity of κ-Car systems were greater in presence of KCl than CaCl2. Unlike κ-Car systems, the gelation of α-Car in presence of KCl was observed without syneresis. Thus, the position of sulfate group on the carrabiose determines the importance of counterion valency too. The α-Car could be a good alternative to κ-Car to reduce the syneresis effects.

Carrageenans are sulfated galactans densely packed in the cell wall of numerous red algae (Rhodophyta). The most abundant in algae and the most exploited industrially for their gelling properties are the kappa-(kappa) and the iota-(iota)-carrageenans which the carrabiose repetition units are substituted by one or two sulfate ester groups. These polyanionic polysaccharides represent an important marine biomass and are carbon source for a number of marine bacteria. One polysaccharide utilization loci, a cluster of genes co-localized in the genome of the marine strain Paraglaciecola atlantica T6c, encompasses several glycoside hydrolases and sulfatases dedicated to the degradation of carrageenan. In addition to one iota-carrageenase and three carrageenan sulfatases, already char-acterized, we discovered the first endo-alpha-carrageenase and an exo-alpha-carrageenase sulfatase which allowed describing an alternate way to the degradation of iota-carrageenan without iota-carrageenase. alpha-Carrageenan didn't make gels in presence of salt suggesting that desulfation of iota-carrageenan probably helps to solubilize the gelling carrageenan. The degradation of the kappa-carrageenan fraction of hybrid kappa-/iota-carrageenan was also obtained without kappa-carrageenase.

Carrageenans are sulfated galactans densely packed in the cell wall of numerous red algae (Rhodophyta). The most abundant in algae and the most exploited industrially for their gelling properties are the κ- (kappa) and the ι- (iota)-carrageenans which the carrabiose repetition units are substituted by one or two sulfate ester groups. These polyanionic polysaccharides represent an important marine biomass and are carbon source for a number of marine bacteria. One polysaccharide utilization loci, a cluster of genes co-localized in the genome of the marine strain Paraglaciecola atlantica T6c, encompasses several glycoside hydrolases and sulfatases dedicated to the degradation of carrageenan. In addition to one ι-carrageenase and three carrageenan sulfatases, already characterized, we discovered the first endo-α-carrageenase and an exo-α-carrageenase sulfatase which allowed describing an alternate way to the degradation of ι-carrageenan without ι-carrageenase. α-Carrageenan didn't make gels in presence of salt suggesting that desulfation of ι-carrageenan probably helps to solubilize the gelling carrageenan. The degradation of the κ-carrageenan fraction of hybrid κ-/ι-carrageenan was also obtained without κ-carrageenase.

Noctoc commune is a cyanobacterium living in various and extreme environments. Its ability to survive in desert, on ice or high altitude is explained by its exceptional metabolism and its capacity to resist to desiccation. N. commune cells are embedded in a gelatinous matrix made of polysaccharides which fixes water and participates in maintaining the cells in hydrated conditions. The structure of the polysaccharide of N. commune harvested in Saint Martin d’Uriage (France) and the oligosaccharides obtained after its enzymatic degradation were determined. The repeating unit of the main chain is a tetra-saccharide: [→4)-β-D-Glcp-(1 → 4)-β-D-Xylp-(1 → 4)-β-D-Glcp-(1 → 4)-α-D-Galp-(1→], branched at position 6 of a glucose residue by a β-linked pyruvated glucuronic acid residue. About 30% of the Xylp residues were branched with a Xylf residue. Comparison of this structure with the polysaccharides secreted by other Nostoc species and strains suggest a strong selection pressure on the structure in agreement with its important biological role.

Diabolican, or HE800, is an exopolysaccharide secreted by the non-pathogenic Gram-negative marine bacterium Vibrio diabolicus (CNCM I-1629). This polysaccharide was enzymatically degraded by the Bacteroides cellulosilyticus WH2 hyaluronan lyase. The end products were purified by size-exclusion chromatography and their structures were analyzed in depth by nuclear magnetic resonance (NMR). The oligosaccharide structures confirmed the possible site of cleavage of the enzyme showing plasticity in the substrate recognitions. The production of glycosaminoglycan-mimetic oligosaccharides of defined molecular weight and structure opens new perspectives in the valorization of the marine polysaccharide diabolican.

β-Mannans are a heterogeneous group of polysaccharides with a common main chain of β-1,4-linked mannopyranoside residues. The cleavage of β-mannan chains is catalyzed by glycoside hydrolases called β-mannanases. In the CAZy database, β-mannanases are grouped by sequence similarity in families GH5, GH26, GH113 and GH134. Family GH113 has been under-explored so far with six enzymes characterized, all from the Firmicutes phylum. We undertook the functional characterization of 14 enzymes from a selection of 31 covering the diversity of the family GH113. Our observations suggest that GH113 is a family with specificity towards mannans, with variations in the product profiles and modes of action. We were able to assign mannanase and mannosidase activities to four out of the five clades of the family, increasing by 200% the number of characterized GH113 members, and expanding the toolbox for fine-tuning of mannooligosaccharides.

The polysaccharide lyase family 6 (PL6) represents one of the 41 polysaccharide lyase families classified in the CAZy database with the vast majority of its members being alginate lyases grouped into three subfamilies, PL6_1-3. To decipher the mode of recognition and action of the enzymes belonging to subfamily PL6_1, we solved the crystal structures of Pedsa0632, Patl3640, Pedsa3628 and Pedsa3807, which all show different substrate specificities and mode of action (endo-/exolyase). Thorough exploration of the structures of Pedsa0632 and Patl3640 in complex with their substrates as well as docking experiments confirms that the conserved residues in subsites -1 to +3 of the catalytic site form a common platform that can accommodate various types of alginate in a very similar manner but with a series of original adaptations bringing them their specificities of action. From comparative studies with existing structures of PL6_1 alginate lyases, we observe that in the right-handed parallel β-helix fold shared by all these enzymes, the substrate-binding site harbors the same overall conserved structures and organization. Despite this apparent similarity, it appears that members of the PL6_1 subfamily specifically accommodate and catalyze the degradation of different alginates suggesting that this common platform is actually a highly adaptable and specific tool.

New insights into the structure of the hybrid κ/β-carrageenan (κ/β-CRG) of the red alga Tichocarpus crinitus have been obtained. Carrageenan oligosaccharides were prepared through the chemical and enzymatic depolymerization of κ/β-CRG with κ-carrageenase and its the enzyme-resistant fraction. The composition and distribution of the repetition units of κ/β- CRG were investigated by using the negative ion tandem MALDI-TOFMS and ESIMS method, which made it possible to prove and characterize the hybrid structure of this polysaccharide. An analysis revealed the blockwise distribution of the long β-blocks along the polysaccharide chain, with the inclusion of κ/β, μ/ν-blocks and some ι-blocks. Furthermore, the desulfated κ/β-CRG was shown to contain of –G–D– repeating units up to 3.5 kDa. Previous studies have demonstrated that CRGs suppress the replication of several viruses. Here, we established that κ/β-CRG and its oligosaccharides significantly inhibit the transduction efficiency of replication-defective lentiviral particles pseudotyped with the envelope proteins of three different viruses. We found that the polysaccharide and its oligosaccharides strongly reduced the transduction efficiency of lentiviral particles pseudotyped with GP160—the envelope protein of the human immunodeficiency virus HIV-1—when added to T-lymphocyte Jurkat cells. The CRG oligosaccharides displayed significantly higher antiviral activity.

Vibrio alginolyticus (CNCM I-5035) secretes an exopolysaccharide used as ingredient in cosmetic industry under the trademark Epidermist 4.0TM. It is appreciated for its ability to improve the physical and chemical barrier functions of the skin by notably increasing the keratinocyte differentiation and epidermal renewal. Composition analyses and in depth characterization of the polysaccharides as well as oligosaccharides obtained by mild acid hydrolyses revealed that it was composed of a repetition unit of three residues: d-galactose (d-Gal), d-N-acetylglucosamine (GlcNAc) and l-N-acetylguluronic acid, of which 30% (M/M) was acetylated in position 3. The complete structure of the polysaccharide was resolved giving the repetition unit: [→3)-α-d-Gal-(1→4)-α-l-GulNAcA/α-l-3OAc-GulNAcA-(1→4)-β-d-GlcNAc-(1→].

To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a β-xylosidase, a α-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end.

Over the last two decades, the number of gene/protein sequences gleaned from sequencing projects of individual genomes and environmental DNA has grown exponentially. Only a tiny fraction of these predicted proteins has been experimentally characterized, and the function of most proteins remains hypothetical or only predicted based on sequence similarity. Despite the development of postgenomic methods, such as transcriptomics, proteomics, and metabolomics, the assignment of function to protein sequences remains one of the main challenges in modern biology. As in all classes of proteins, the growing number of predicted carbohydrate-active enzymes (CAZymes) has not been accompanied by a systematic and accurate attribution of function. Taking advantage of the CAZy database, which groups CAZymes into families and subfamilies based on amino acid similarities, we recombinantly produced 564 proteins selected from subfamilies without any biochemically characterized representatives, from distant relatives of characterized enzymes and from nonclassified proteins that show little similarity with known CAZymes. Screening these proteins for activity on a wide collection of carbohydrate substrates led to the discovery of 13 CAZyme families (two ofwhich were also discovered by others during the course of our work), revealed three previously unknown substrate specificities, and assigned a function to 25 subfamilies.

Vibrio alginolyticus (CNCM I-4151) secretes an exopolysaccharide whose carbohydrate backbone is decorated with amino acids, likely conferring its properties that are appreciated in cosmetics. Here, the secreted polysaccharide of another strain of V. alginolyticus (CNCM I-5034) was characterized by chromatography and one- and two-dimensional NMR spectroscopy experiments. The structure was resolved and shows that the carbohydrate backbone is made of four residues: D-galactose (Gal), D-galacturonic acid (GalA) D-N-acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA), forming a tetrasaccharide repetition unit [→4)-β-d-GlcA-(1→3)-α-d-Gal-(1→3)-α-d-GalA-(1→3)-β-GlcNAc(1→]. GlcA is derivatized with a lactate group giving ‘nosturonic acid’, and GalA is decorated with the amino acid alanine.

In bacteria from the phylum Bacteroidetes, the genes coding for enzymes involved in polysaccharide degradation are often colocalized and coregulated in so-called "polysaccharide utilization loci" (PULs). PULs dedicated to the degradation of marine polysaccharides (e.g. laminaran, ulvan, alginate and porphyran) have been characterized in marine bacteria. Interestingly, the gut microbiome of Japanese individuals acquired, by lateral transfer from marine bacteria, the genes involved in the breakdown of porphyran, the cell wall polysaccharide of the red seaweed used in maki. Sequence similarity analyses predict that the human gut microbiome also encodes enzymes for the degradation of alginate, the main cell wall polysaccharide of brown algae. We undertook the functional characterization of diverse polysaccharide lyases from family PL17, frequently found in marine bacteria as well as those of human gut bacteria. We demonstrate here that this family is polyspecific. Our phylogenetic analysis of family PL17 reveals that all alginate lyases, which have all the same specificity and mode of action, cluster together in a very distinct subfamily. The alginate lyases found in human gut bacteria group together in a single clade which is rooted deeply in the PL17 tree. These enzymes were found in PULs containing PL6 enzymes, which also clustered together in the phylogenetic tree of PL6. Together, biochemical and bioinformatics analyses suggest that acquisition of this system appears ancient and, because only traces of two successful transfers were detected upon inspection of PL6 and PL17 families, the pace of acquisition of marine polysaccharide degradation system is probably very slow.

Ulvans are the sulfated cell wall polysaccharide present in marine green algae. Ulvan polysaccharides are composed mainly of 3‐sulfated rhamnose, glucuronic acid, iduronic acid and xylose. The sulfation pattern in ulvan resemble glycosaminoglycans in vertebrates. The structural features of ulvan make it a good candidate for variety of industrial applications in agriculture, food, pharmaceutical, chemical, and biomaterials industries. Identification of ulvan‐degrading microorganism and the corresponding enzymes will increase the potential application of this highly abundant naturally occurring polysaccharide.Bacterial microbiomes associated with the green algae contain enzymes to degrade ulvan by a lytic β‐elimination mechanism. Genome sequencing projects lead to the identification of many such ulvan degrading enzymes from several bacteria Nonlabens ulvanivorans, Pseudoalteromonas sp. strain PLSV, Alteromonas sp. strain LOR and LTR. Using X‐ray crystallography and enzyme activity assays, we solved the structure and biochemically characterized three ulvan lyases, PLSV3936, LOR107 and NLR48. All three enzymes share very low sequence identity and act differentially on substrate.Despite their low sequence identity, PLSV3936 and LOR107 share the same 7‐bladed β propeller fold. However, the complex structure with the bound tetrasaccharide substrate reveals the difference in the active site and mode of cleavage. Whereas, NLR48 has a β jelly roll scaffold. Complex structure of NLR48 with tetrasaccharide substrate suggest that NLR48 appears to utilize lysine and tyrosine as catalytic residues and the substrate acidic group is neutralized by a glutamine residue. Our results expand the information about ulvan degrading enzymes to potentiate the use of ulvan polysaccharide.Support or Funding InformationThe financial support was provided by a grant from the Natural Science and Engineering Research Council of CanadaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ulvan is a complex sulfated polysaccharide present in the cell wall of green algae of the genus Ulva (Chlorophyta). The first ulvan-degrading polysaccharide lyases were identified several years ago, and more were discovered through genome sequencing of marine bacteria. Ulvan lyases are now grouped in three polysaccharide lyase (PL) families in the CAZy database, PL24, PL25, and PL28. The recently determined structures of the representative lyases from families PL24 and PL25 show that they adopt a seven-bladed β-propeller fold and utilize the His/Tyr catalytic mechanism. No structural information is yet available for PL28 ulvan lyases. NLR48 from Nonlabens ulvanivorans belongs to PL28 together with its close paralog, NLR42. Biochemical studies of NLR42 have revealed that it can cleave ulvan next to both uronic acid epimers. We report the crystal structure of ulvan lyase NLR48 at 1.9-Å resolution. It has a β-jelly roll fold with an extended, deep, and positively charged substrate-binding cleft. Putative active-site residues were identified from the sequence conservation pattern, and their role was confirmed by site-directed mutagenesis. The structure of an inactive K162M mutant with a tetrasaccharide substrate showed the substrate occupying the “−” subsites. Comparison with lyases from other PL families with β-jelly roll folds supported assignment of the active site and explained its ability to degrade ulvan next to either epimer of uronic acid. NLR48 contains the His/Tyr catalytic machinery with Lys162 and Tyr281 playing the catalytic base/acid roles.

Ulvan is a major cell wall component of green algae of the genus Ulva, and some marine bacteria encode enzymes that can degrade this polysaccharide. The first ulvan-degrading lyases have been recently characterized, and several putative ulvan lyases have been recombinantly expressed, confirmed as ulvan lyases, and partially characterized. Two families of ulvan-degrading lyases, PL24 and PL25, have recently been established. The PL24 lyase LOR_107 from the bacterial Alteromonadales sp. strain LOR degrades ulvan endolytically, cleaving the bond at the C4 of a glucuronic acid. However, the mechanism and LOR_107 structural features involved are unknown. We present here the crystal structure of LOR_107, representing the first PL24 family structure. We found that LOR_107 adopts a seven-bladed β-propeller fold with a deep canyon on one side of the protein. Comparative sequence analysis revealed a cluster of conserved residues within this canyon, and site-directed mutagenesis disclosed several residues essential for catalysis. We also found that LOR_107 uses the His/Tyr catalytic mechanism, common to several PL families. We captured a tetrasaccharide substrate in the structures of two inactive mutants, which indicated a two-step binding event, with the first substrate interaction near the top of the canyon coordinated by Arg320, followed by sliding of the substrate into the canyon toward the active-site residues. Surprisingly, the LOR_107 structure was very similar to that of the PL25 family PLSV_3936, despite only ∼14% sequence identity between the two enzymes. On the basis of our structural and mutational analyses, we propose a catalytic mechanism for LOR_107 that differs from the typical His/Tyr mechanism.