Microbial-host-isozyme analyses reveal microbial DPP4 as a potential antidiabetic target.

A mechanistic understanding of how microbial proteins affect the host could yield deeper insights into gut microbiota-host cross-talk. We developed an enzyme activity-screening platform to investigate how gut microbiota-derived enzymes might influence host physiology. We discovered that dipeptidyl peptidase 4 (DPP4) is expressed by specific bacterial taxa of the microbiota. Microbial DPP4 was able to decrease the active glucagon like peptide-1 (GLP-1) and disrupt glucose metabolism in mice with a leaky gut. Furthermore, the current drugs targeting human DPP4, including sitagliptin, had little effect on microbial DPP4. Using high-throughput screening, we identified daurisoline-d4 (Dau-d4) as a selective microbial DPP4 inhibitor that improves glucose tolerance in diabetic mice.

NMR Analyses of the Enzymatic Degradation End-Products of Diabolican: The Secreted EPS of Vibrio diabolicus CNCM I-1629.

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.

Molecular and Biochemical Analyses of a Novel Trifunctional Endoxylanase/Endoglucanase/Feruloyl Esterase from the Human Colonic Bacterium Bacteroides intestinalis DSM 17393.

A novel enzyme Bi76 comprising GH10, E_set_Esterase_N, and CE1 modules was identified, with the highest homology (62.9%) with a bifunctional endoxylanase/feruloyl esterase among characterized enzymes. Interestingly, Bi76 hydrolyzed glucan substrates besides xylans and feruloylated substrates, suggesting that it is the first characterized trifunctional endoxylanase/endoglucanase/feruloyl esterase. Analyses of truncation variants revealed that GH10 and E_set_Esterase_N + CE1 modules encoded endoxylanase/endoglucanase and feruloyl esterase activities, respectively. Synergism analyses indicated that endoxylanase, α-l-arabinofuranosidase, and feruloyl esterase acted cooperatively in releasing ferulic acid (FA) and xylooligosaccharides from feruloylated arabinoxylan. The interdomain synergism of Bi76 overmatched the intermolecular synergism of TM1 and TM2. Importantly, Bi76 exhibited good capacity in producing FA, releasing 5.20, 4.38, 2.12, 1.35, 0.46, and 0.19 mg/g from corn bran, corn cob, wheat bran, corn stover, rice husk, and rice bran, respectively. This study expands the trifunctional endoxylanase/endoglucanase/feruloyl esterase repertoire and demonstrates the great potential of Bi76 in agricultural residue utilization.

Multi-omics analyses of the ulcerative colitis gut microbiome link Bacteroides vulgatus proteases with disease severity.

Ulcerative colitis (UC) is driven by disruptions in host-microbiota homoeostasis, but current treatments exclusively target host inflammatory pathways. To understand how host-microbiota interactions become disrupted in UC, we collected and analysed six faecal- or serum-based omic datasets (metaproteomic, metabolomic, metagenomic, metapeptidomic and amplicon sequencing profiles of faecal samples and proteomic profiles of serum samples) from 40 UC patients at a single inflammatory bowel disease centre, as well as various clinical, endoscopic and histologic measures of disease activity. A validation cohort of 210 samples (73 UC, 117 Crohn's disease, 20 healthy controls) was collected and analysed separately and independently. Data integration across both cohorts showed that a subset of the clinically active UC patients had an overabundance of proteases that originated from the bacterium Bacteroides vulgatus. To test whether B. vulgatus proteases contribute to UC disease activity, we first profiled B. vulgatus proteases found in patients and bacterial cultures. Use of a broad-spectrum protease inhibitor improved B. vulgatus-induced barrier dysfunction in vitro, and prevented colitis in B. vulgatus monocolonized, IL10-deficient mice. Furthermore, transplantation of faeces from UC patients with a high abundance of B. vulgatus proteases into germfree mice induced colitis dependent on protease activity. These results, stemming from a multi-omics approach, improve understanding of functional microbiota alterations that drive UC and provide a resource for identifying other pathways that could be inhibited as a strategy to treat this disease.

A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes.

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species-Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked ß1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

A single sulfatase is required to access colonic mucin by a gut bacterium.

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel disease3.

Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB.

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.

BdPUL12 depolymerizes ß-mannan-like glycans into mannooligosaccharides and mannose, which serve as carbon sources for Bacteroides dorei and gut probiotics.

Symbiotic bacteria, including members of the Bacteroides genus, are known to digest dietary fibers in the gastrointestinal tract. The metabolism of complex carbohydrates is restricted to a specified subset of species and is likely orchestrated by polysaccharide utilization loci (PULs) in these microorganisms. ß-Mannans are plant cell wall polysaccharides that are commonly found in human nutrients. Here, we report the structural basis of a PUL cluster, BdPUL12, which controls ß-mannan-like glycan catabolism in Bacteroides dorei. Detailed biochemical characterization and targeted gene disruption studies demonstrated that a key glycoside hydrolase, BdP12GH26, performs the initial attack on galactomannan or glucomannan likely via an endo-acting mode, generating mannooligosaccharides and mannose. Importantly, coculture assays showed that the B. dorei promoted the proliferation of Lactobacillus helveticus and Bifidobacterium adolescentis, likely by sharing mannooligosaccharides and mannose with these gut probiotics. Our findings provide new insights into carbohydrate metabolism in gut-inhabiting bacteria and lay a foundation for novel probiotic development.

Sulfation of Arabinogalactan Proteins Confers Privileged Nutrient Status to Bacteroides plebeius.

The human gut microbiota (HGM) contributes to the physiology and health of its host. The health benefits provided by dietary manipulation of the HGM require knowledge of how glycans, the major nutrients available to this ecosystem, are metabolized. Arabinogalactan proteins (AGPs) are a ubiquitous feature of plant polysaccharides available to the HGM. Although the galactan backbone and galactooligosaccharide side chains of AGPs are conserved, the decorations of these structures are highly variable. Here, we tested the hypothesis that these variations in arabinogalactan decoration provide a selection mechanism for specific Bacteroides species within the HGM. The data showed that only a single bacterium, B. plebeius, grew on red wine AGP (Wi-AGP) and seaweed AGP (SW-AGP) in mono- or mixed culture. Wi-AGP thus acts as a privileged nutrient for a Bacteroides species within the HGM that utilizes marine and terrestrial plant glycans. The B. plebeius polysaccharide utilization loci (PULs) upregulated by AGPs encoded a polysaccharide lyase, located in the enzyme family GH145, which hydrolyzed Rha-Glc linkages in Wi-AGP. Further analysis of GH145 identified an enzyme with two active sites that displayed glycoside hydrolase and lyase activities, respectively, which conferred substrate flexibility for different AGPs. The AGP-degrading apparatus of B. plebeius also contained a sulfatase, BpS1_8, active on SW-AGP and Wi-AGP, which played a pivotal role in the utilization of these glycans by the bacterium. BpS1_8 enabled other Bacteroides species to access the sulfated AGPs, providing a route to introducing privileged nutrient utilization into probiotic and commensal organisms that could improve human health. IMPORTANCE Dietary manipulation of the HGM requires knowledge of how glycans available to this ecosystem are metabolized. The variable structures that decorate the core component of plant AGPs may influence their utilization by specific organisms within the HGM. Here, we evaluated the ability of Bacteroides species to utilize a marine and terrestrial AGP. The data showed that a single bacterium, B. plebeius, grew on Wi-AGP and SW-AGP in mono- or mixed culture. Wi-AGP is thus a privileged nutrient for a Bacteroides species that utilizes marine and terrestrial plant glycans. A key component of the AGP-degrading apparatus of B. plebeius is a sulfatase that conferred the ability of the bacterium to utilize these glycans. The enzyme enabled other Bacteroides species to access the sulfated AGPs, providing a route to introducing privileged nutrient utilization into probiotic and commensal organisms that could improve human health.

Metabolic and enzymatic elucidation of cooperative degradation of red seaweed agarose by two human gut bacteria.

Various health beneficial outcomes associated with red seaweeds, especially their polysaccharides, have been claimed, but the molecular pathway of how red seaweed polysaccharides are degraded and utilized by cooperative actions of human gut bacteria has not been elucidated. Here, we investigated the enzymatic and metabolic cooperation between two human gut symbionts, Bacteroides plebeius and Bifidobacterium longum ssp. infantis, with regard to the degradation of agarose, the main carbohydrate of red seaweed. More specifically, B. plebeius initially decomposed agarose into agarotriose by the actions of the enzymes belonging to glycoside hydrolase (GH) families 16 and 117 (i.e., BpGH16A and BpGH117) located in the polysaccharide utilization locus, a specific gene cluster for red seaweed carbohydrates. Then, B. infantis extracted energy from agarotriose by the actions of two agarolytic ß-galactosidases (i.e., Bga42A and Bga2A) and produced neoagarobiose. B. plebeius ultimately acted on neoagarobiose by BpGH117, resulting in the production of 3,6-anhydro-L-galactose, a monomeric sugar possessing anti-inflammatory activity. Our discovery of the cooperative actions of the two human gut symbionts on agarose degradation and the identification of the related enzyme genes and metabolic intermediates generated during the metabolic processes provide a molecular basis for agarose degradation by gut bacteria.

Characterization of Neoagarooligosaccharide Hydrolase BpGH117 from a Human Gut Bacterium Bacteroides plebeius.

α-Neoagarobiose (NAB)/neoagarooligosaccharide (NAO) hydrolase plays an important role as an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase in agarose utilization. Agarose is an abundant polysaccharide found in red seaweeds, comprising 3,6-anhydro-L-galactose (AHG) and D-galactose residues. Unlike agarose degradation, which has been reported in marine microbes, recent metagenomic analysis of Bacteroides plebeius, a human gut bacterium, revealed the presence of genes encoding enzymes involved in agarose degradation, including α-NAB/NAO hydrolase. Among the agarolytic enzymes, BpGH117 has been partially characterized. Here, we characterized the exo-acting α-NAB/NAO hydrolase BpGH117, originating from B. plebeius. The optimal temperature and pH for His-tagged BpGH117 activity were 35 °C and 9.0, respectively, indicative of its unique origin. His-tagged BpGH117 was thermostable up to 35 °C, and the enzyme activity was maintained at 80% of the initial activity at a pre-incubation temperature of 40 °C for 120 min. Km and Vmax values for NAB were 30.22 mM and 54.84 U/mg, respectively, and kcat/Km was 2.65 s-1 mM-1. These results suggest that His-tagged BpGH117 can be used for producing bioactive products such as AHG and agarotriose from agarose efficiently.

Orthogonal Active-Site Labels for Mixed-Linkage endo-ß-Glucanases.

Small molecule irreversible inhibitors are valuable tools for determining catalytically important active-site residues and revealing key details of the specificity, structure, and function of glycoside hydrolases (GHs). ß-glucans that contain backbone ß(1,3) linkages are widespread in nature, e.g., mixed-linkage ß(1,3)/ß(1,4)-glucans in the cell walls of higher plants and ß(1,3)glucans in yeasts and algae. Commensurate with this ubiquity, a large diversity of mixed-linkage endoglucanases (MLGases, EC 3.2.1.73) and endo-ß(1,3)-glucanases (laminarinases, EC 3.2.1.39 and EC 3.2.1.6) have evolved to specifically hydrolyze these polysaccharides, respectively, in environmental niches including the human gut. To facilitate biochemical and structural analysis of these GHs, with a focus on MLGases, we present here the facile chemo-enzymatic synthesis of a library of active-site-directed enzyme inhibitors based on mixed-linkage oligosaccharide scaffolds and N-bromoacetylglycosylamine or 2-fluoro-2-deoxyglycoside warheads. The effectiveness and irreversibility of these inhibitors were tested with exemplar MLGases and an endo-ß(1,3)-glucanase. Notably, determination of inhibitor-bound crystal structures of a human-gut microbial MLGase from Glycoside Hydrolase Family 16 revealed the orthogonal labeling of the nucleophile and catalytic acid/base residues with homologous 2-fluoro-2-deoxyglycoside and N-bromoacetylglycosylamine inhibitors, respectively. We anticipate that the selectivity of these inhibitors will continue to enable the structural and mechanistic analyses of ß-glucanases from diverse sources and protein families.

Structural basis for the exolytic activity of polysaccharide lyase family 6 alginate lyase BcAlyPL6 from human gut microbe Bacteroides clarus.

Alginate is the structural polysaccharide of the cell wall of brown algae, which is an important carbon source for marine life. The depolymerization of alginate is dependent on alginate lyases. Recent studies showed that the alginate utilization ability had been obtained by human gut microbes. In contrast to the great number of studies on alginate lyases from marine/soil organisms, studies on alginate lyases from gut microbes are still limited. Here, the structure of a polysaccharide lyase family 6 (PL6) alginate lyase from human gut microbe Bacteroides clarus was solved by X-ray crystallography, which represents the cluster of two-domain PL6 alginate lyases from Bacteroidetes. Similar with the two-domain alginate lyase AlyGC originated from marine bacterium, both the N terminal domain (NTD) and C terminal domain (CTD) of BcAlyPL6 show right-handed parallel ß-helix fold. However, unlike AlyGC, which forms a homodimer, BcAlyPL6 functions as a monomer. Biochemical analysis indicates that the substrate binding affinity is mainly contributed by the NTD while the CTD of BcAlyPL6 is involved in the formation of -1 subsite, which is essential for substrate turnover rate. Furthermore, CTD is involved in shaping a closed catalytic pocket, and deletion of it leads to increased activity towards highly polymerized substrate. Structure comparison of PL6 family alginate lyases implies that the linkers of two-domain alginate lyases might have evolutionary relationship with the N/C terminal extension of single-domain lyases.

Degradation of complex arabinoxylans by human colonic Bacteroidetes.

Some Bacteroidetes and other human colonic bacteria can degrade arabinoxylans, common polysaccharides found in dietary fiber. Previous work has identified gene clusters (polysaccharide-utilization loci, PULs) for degradation of simple arabinoxylans. However, the degradation of complex arabinoxylans (containing side chains such as ferulic acid, a phenolic compound) is poorly understood. Here, we identify a PUL that encodes multiple esterases for degradation of complex arabinoxylans in Bacteroides species. The PUL is specifically upregulated in the presence of complex arabinoxylans. We characterize some of the esterases biochemically and structurally, and show that they release ferulic acid from complex arabinoxylans. Growth of four different colonic Bacteroidetes members, including Bacteroides intestinalis, on complex arabinoxylans results in accumulation of ferulic acid, a compound known to have antioxidative and immunomodulatory properties.

A highly active heparinase I from Bacteroides cellulosilyticus: Cloning, high level expression, and molecular characterization.

As one of the most extensively studied glycosaminoglycan lyases, heparinase I has been used in producing low or ultra-low molecular weight heparin. Its' important applications are to neutralize the heparin in human blood and analyze heparin structure in the clinic. However, the low productivity and activity of the enzyme have greatly hindered its applications. In this study, a novel Hep-I from Bacteroides cellulosilyticus (BcHep-I) was successfully cloned and heterologously expressed in E. coli BL21 (DE3) as a soluble protein. The molecular mass and isoelectric point (pI) of the enzyme are 44.42 kDa and 9.02, respectively. And the characterization of BcHep-I after purified with Ni-NTA affinity chromatography suggested that it is a mesophilic enzyme. BcHep-I can be activated by 1 mM Ca2+, Mg2+, and Mn2+, while severely inhibited by Zn2+, Co2+, and EDTA. The specific activity of the enzyme was 738.3 U·mg-1 which is the highest activity ever reported. The Km and Vmax were calculated as 0.17 mg·mL-1 and 740.58 U·mg-1, respectively. Besides, the half-life of 300 min at 30°C showed BcHep-I has practical applications. Homology modeling and substrate docking revealed that Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 were mainly involved in the substrate binding of Hep-I, and 11 hydrogen bonds were formed between heparin and the enzyme. These results indicated that BcHep-I with high activity has great potential applications in the industrial production of heparin, especially in the clinic to neutralize heparin.

Linear and branched ß-Glucans degrading enzymes from versatile Bacteroides uniformis JCM 13288T and their roles in cooperation with gut bacteria.

ß-glucans are the dietary nutrients present in oats, barley, algae, and mushrooms. The macromolecules are well known for their immune-modulatory activity; however, how the human gut bacteria digest them is vaguely understood. In this study, Bacteroides uniformis JCM 13288 T was found to grow on laminarin, pustulan, and porphyran. We sequenced the genome of the strain, which was about 5.05 megabase pairs and contained 4868 protein-coding genes. On the basis of growth patterns of the bacterium, two putative polysaccharide utilization loci for ß-glucans were identified from the genome, and associated four putative genes were cloned, expressed, purified, and characterized. Three glycoside hydrolases (GHs) that were endo-acting enzymes (BuGH16, BuGH30, and BuGH158), and one which was an exo-acting (BuGH3) enzyme. The BuGH3, BuGH16, and BuGH158 can cleave linear exo/endo- ß- 1-3 linkages while BuGH30 can digest endo- ß- 1-6 linkages. BuGH30 and BuGH158 were further explored for their roles in digesting ß- glucans and generation of oligosaccharides, respectively. The BuGH30 predominately found to cleave long chain ß- 1-6 linked glucans, and obtained final product was gentiobiose. The BuGH158 used for producing oligosaccharides varying from degree of polymerization 2 to 7 from soluble curdlan. We demonstrated that these oligosaccharides can be utilized by gut bacteria, which either did not grow or poorly grew on laminarin. Thus, B. uniformis JCM 13288 T is not only capable of utilizing ß-glucans but also shares these glycans with human gut bacteria for potentially maintaining the gut microbial homeostasis.

A carbohydrate-active enzyme (CAZy) profile links successful metabolic specialization of Prevotella to its abundance in gut microbiota.

Gut microbiota participates in diverse metabolic and homeostatic functions related to health and well-being. Its composition varies between individuals, and depends on factors related to host and microbial communities, which need to adapt to utilize various nutrients present in gut environment. We profiled fecal microbiota in 63 healthy adult individuals using metaproteomics, and focused on microbial CAZy (carbohydrate-active) enzymes involved in glycan foraging. We identified two distinct CAZy profiles, one with many Bacteroides-derived CAZy in more than one-third of subjects (n = 25), and it associated with high abundance of Bacteroides in most subjects. In a smaller subset of donors (n = 8) with dietary parameters similar to others, microbiota showed intense expression of Prevotella-derived CAZy including exo-beta-(1,4)-xylanase, xylan-1,4-beta-xylosidase, alpha-L-arabinofuranosidase and several other CAZy belonging to glycosyl hydrolase families involved in digestion of complex plant-derived polysaccharides. This associated invariably with high abundance of Prevotella in gut microbiota, while in subjects with lower abundance of Prevotella, microbiota showed no Prevotella-derived CAZy. Identification of Bacteroides- and Prevotella-derived CAZy in microbiota proteome and their association with differences in microbiota composition are in evidence of individual variation in metabolic specialization of gut microbes affecting their colonizing competence.

Identification of Protein Recognition Elements within Heparin Chains Using Enzymatic Foot-Printing in Solution and Online SEC/MS.

Understanding molecular mechanisms governing interactions of glycosaminoglycans (such as heparin) with proteins remains challenging due to their enormous structural heterogeneity. Commonly accepted approaches seek to reduce the structural complexity by searching for "binding epitopes" within the limited subsets of short heparin oligomers produced either enzymatically or synthetically. A top-down approach presented in this work seeks to preserve the chemical diversity displayed by heparin by allowing the longer and structurally diverse chains to interact with the client protein. Enzymatic lysis of the protein-bound heparin chains followed by the product analysis using size exclusion chromatography with online mass spectrometry detection (SEC/MS) reveals the oligomers that are protected from lysis due to their tight association with the protein, and enables their characterization (both the oligomer length, and the number of incorporated sulfate and acetyl groups). When applied to a paradigmatic heparin/antithrombin system, the new method generates a series of oligomers with surprisingly distinct sulfation levels. The extent of sulfation of the minimal-length binder (hexamer) is relatively modest yet persistent, consistent with the notion of six sulfate groups being both essential and sufficient for antithrombin binding. However, the masses of longer surviving chains indicate complete sulfation of disaccharides beyond the hexasaccharide core. Molecular dynamics simulations confirm the existence of favorable electrostatic interactions between the high charge-density saccharide residues flanking the "canonical" antithrombin-binding hexasaccharide and the positive patch on the surface of the overall negatively charged protein. Furthermore, electrostatics may rescue the heparin/protein interaction in the absence of the canonical binding element.

Synergy between Cell Surface Glycosidases and Glycan-Binding Proteins Dictates the Utilization of Specific Beta(1,3)-Glucans by Human Gut Bacteroides.

The human gut microbiota (HGM) has far-reaching impacts on human health and nutrition, which are fueled primarily by the metabolism of otherwise indigestible complex carbohydrates commonly known as dietary fiber. However, the molecular basis of the ability of individual taxa of the HGM to address specific dietary glycan structures remains largely unclear. In particular, the utilization of ß(1,3)-glucans, which are widespread in the human diet as yeast, seaweed, and plant cell walls, had not previously been resolved. Through a systems-based approach, here we show that the symbiont Bacteroides uniformis deploys a single, exemplar polysaccharide utilization locus (PUL) to access yeast ß(1,3)-glucan, brown seaweed ß(1,3)-glucan (laminarin), and cereal mixed-linkage ß(1,3)/ß(1,4)-glucan. Combined biochemical, enzymatic, and structural analysis of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) illuminates a concerted molecular system by which B. uniformis recognizes and saccharifies these distinct ß-glucans. Strikingly, the functional characterization of homologous ß(1,3)-glucan utilization loci (1,3GUL) in other Bacteroides further demonstrated that the ability of individual taxa to utilize ß(1,3)-glucan variants and/or ß(1,3)/ß(1,4)-glucans arises combinatorially from the individual specificities of SGBPs and GHs at the cell surface, which feed corresponding signals to periplasmic hybrid two-component sensors (HTCSs) via TonB-dependent transporters (TBDTs). These data reveal the importance of cooperativity in the adaptive evolution of GH and SGBP cohorts to address individual polysaccharide structures. We anticipate that this fine-grained knowledge of PUL function will inform metabolic network analysis and proactive manipulation of the HGM. Indeed, a survey of 2,441 public human metagenomes revealed the international, yet individual-specific, distribution of each 1,3GUL.IMPORTANCEBacteroidetes are a dominant phylum of the human gut microbiota (HGM) that target otherwise indigestible dietary fiber with an arsenal of polysaccharide utilization loci (PULs), each of which is dedicated to the utilization of a specific complex carbohydrate. Here, we provide novel insight into this paradigm through functional characterization of homologous PULs from three autochthonous Bacteroides species, which target the family of dietary ß(1,3)-glucans. Through detailed biochemical and protein structural analysis, we observed an unexpected diversity in the substrate specificity of PUL glycosidases and glycan-binding proteins with regard to ß(1,3)-glucan linkage and branching patterns. In combination, these individual enzyme and protein specificities support taxon-specific growth on individual ß(1,3)-glucans. This detailed metabolic insight, together with a comprehensive survey of individual 1,3GULs across human populations, further expands the fundamental roadmap of the HGM, with potential application to the future development of microbial intervention therapies.

Cloning, expression, and characterization of a novel heparinase I from Bacteroides eggerthii.

Heparinase I (Hep I) specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in preparation of low molecular weight heparin (LMWH). In this work, a novel Hep I from Bacteroides eggerthii VPI T5-42B-1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The enzyme has specific activity of 480 IU·mg-1 at the optimal temperature and pH of 30 °C and pH 7.5, and the Km and Vmax were 3.6 mg·mL-1 and 647.93 U·mg-1, respectively. The Hep I has good stability with t1/2 values of 350 and 60 min at 30 and 37 °C, respectively. And it showed a residual relative activity of 70.8% after 21 days incubation at 4 °C. Substrate docking study revealed that Lys99, Arg101, Gln241, Lys270, Asn275, and Lys292 were mainly involved in the substrate binding of Hep I. The shorter hydrogen bonds formed between heparin and these residues suggested the higher specific activity of BeHep I. And the minimum conformational entropy value of 756 J·K-1 provides an evidence for the improved stability of this enzyme. This Hep I could be of interest in the industrial preparation of LMWH for its high specific activity and good stability.