Structural Identity of Galactooligosaccharide Molecules Selectively Utilized by Single Cultures of Probiotic Bacterial Strains.

Various ß-galactosidase enzymes catalyze the trans-glycosylation reaction with lactose. The resulting galactooligosaccharide (GOS) mixtures are widely used in infant nutrition to stimulate growth of beneficial gut bacteria. GOS consists mainly of compounds with a degree of polymerization (DP) varying from 2-8 and with diverse glycosidic linkages. In recent years, we have elucidated in detail the composition of several commercial GOS mixtures in terms of DP and the structural identity of the individual compounds. In this work, 13 (single) probiotic strains of gut bacteria, belonging to 11 different species, were grown to stationary phase with a Vivinal GOS-derived sample purified to remove lactose and monosaccharides (pGOS). Growth among the probiotic strains varied strongly between 30 and 100% of OD600nm relative to positive controls with glucose. By identifying the components of the pGOS mixture that remain after growth, we showed that strains varied in their consumption of specific GOS compounds. All strains commonly used most of the GOS DP2 pool. Lactobacillus salivarius W57 also utilized the DP3 branched compound ß-d-Galp-(1 → 4)-[ß-d-Galp-(1 → 2)]-d-Glc. Bifidobacterial strains tended to use GOS with higher DP and branching than lactobacilli; Bifidobacterium breve DSM 20091, Lactobacillus acidophilus W37, and Bifidobacterium infantis DSM 20088 were exceptional in using 38, 36, and 35 compounds, respectively, out of the 40 different structures identified in pGOS. We correlated these bacterial GOS consumption profiles with their genomic information and were able to relate metabolic activity with the presence of genome-encoded transporters and carbohydrate-active enzymes. These detailed insights may support the design of synbiotic combinations pairing probiotic bacterial strains with GOS compounds that specifically stimulate their growth. Such synbiotic combinations may be of interest in food/feed and/or pharmacy/medicine applications.

Structural and functional characterization of a family GH53 ß-1,4-galactanase from Bacteroides thetaiotaomicron that facilitates degradation of prebiotic galactooligosaccharides.

Galactooligosaccharides (GOS) are prebiotic compounds synthesized from lactose using bacterial enzymes and are known to stimulate growth of beneficial bifidobacteria in the human colon. Bacteroides thetaiotaomicron is a prominent human colon commensal bacterial species that hydrolyzes GOS using an extracellular Glycosyl Hydrolase (GH) family GH53 endo-galactanase enzyme (BTGH53), releasing galactose-based products for growth. Here we dissect the molecular basis for GOS activity of this B. thetaiotaomicron GH53 endo-galactanase. Elucidation of its X-ray crystal structure revealed that BTGH53 has a relatively open active site cleft which was not observed with the bacterial enzyme from Bacillus licheniformis (BLGAL). BTGH53 acted on GOS with degree of polymerization ≤3 and therefore more closely resembles activity of fungal GH53 enzymes (e.g. Aspergillus aculeatus AAGAL and Meripileus giganteus MGGAL). Probiotic lactobacilli that lack galactan utilization systems constitute a group of bacteria with relevance for a healthy (infant) gut. The strains tested were unable to use GOS ≥ DP3. However, they completely consumed GOS in the presence of BTGH53, resulting in clear stimulation of their extent of growth. The extracellular BTGH53 enzyme thus may play an important role in carbohydrate metabolism in complex microbial environments such as the human colon. It also may find application for the development of synergistic synbiotics.

Cross-Feeding among Probiotic Bacterial Strains on Prebiotic Inulin Involves the Extracellular exo-Inulinase of Lactobacillus paracasei Strain W20.

Probiotic gut bacteria employ specific metabolic pathways to degrade dietary carbohydrates beyond the capabilities of their human host. Here, we report how individual commercial probiotic strains degrade prebiotic (inulin type) fructans. First, a structural analysis of commercial fructose oligosaccharide-inulin samples was performed. These ß-(2-1)-fructans differ in termination by either glucose (GF) or fructose (FF) residues, with a broad variation in the degrees of polymerization (DPs). The growth of individual probiotic bacteria on short-chain inulin (sc-inulin) (Frutafit CLR), a ß-(2-1)-fructan (DP 2 to DP 40), was studied. Lactobacillus salivarius W57 and other bacteria grew relatively poorly on sc-inulin, with only fractions of DP 3 and DP 5 utilized, reflecting uptake via specific transport systems followed by intracellular metabolism. Lactobacillus paracasei subsp. paracasei W20 completely used all sc-inulin components, employing an extracellular exo-inulinase enzyme (glycoside hydrolase family GH32 [LpGH32], also found in other strains of this species); the purified enzyme converted high-DP compounds into fructose, sucrose, 1-kestose, and F2 (inulobiose). The cocultivation of L. salivarius W57 and L. paracasei W20 on sc-inulin resulted in cross-feeding of the former by the latter, supported by this extracellular exo-inulinase. The extent of cross-feeding depended on the type of fructan, i.e., the GF type (clearly stimulating) versus the FF type (relatively low stimulus), and on fructan chain length, since relatively low-DP ß-(2-1)-fructans contain a relatively high content of GF-type molecules, thus resulting in higher concentrations of GF-type DP 2 to DP 3 degradation products. The results provide an example of how in vivo cross-feeding on prebiotic ß-(2-1)-fructans may occur among probiotic lactobacilli.IMPORTANCE The human gut microbial community is associated strongly with host physiology and human diseases. This observation has prompted research on pre- and probiotics, two concepts enabling specific changes in the composition of the human gut microbiome that result in beneficial effects for the host. Here, we show how fructooligosaccharide-inulin prebiotics are fermented by commercial probiotic bacterial strains involving specific sets of enzymes and transporters. Cross-feeding strains such as Lactobacillus paracasei W20 may thus act as keystone strains in the degradation of prebiotic inulin in the human gut, and this strain-exo-inulinase combination may be used in commercial Lactobacillus-inulin synbiotics.

Draft Genome Sequence of Lactobacillus reuteri 121, a Source of α-Glucan and ß-Fructan Exopolysaccharides.

The probiotic bacterium Lactobacillus reuteri 121 is a well-known producer of diverse homoexopolysaccharides (α-glucans and ß-fructans) from sucrose and maltodextrins/starches of interest for food applications. Here, we report the draft genome sequence of this strain, with a focus on carbohydrate-active enzymes.

Prebiotic galactooligosaccharides activate mucin and pectic galactan utilization pathways in the human gut symbiont Bacteroides thetaiotaomicron.

Galactooligosaccharides (GOS) are prebiotic carbohydrates that impart changes in the gut bacterial composition of formula-fed infants to more closely resemble that of breast-fed infants. Consuming human milk oligosaccharides (HMOs) provides specific bacterial strains with an advantage for colonizing the infant intestine. These same effects are seen in infants after GOS consumption, however GOS are very complex mixtures and the underlying molecular mechanisms of how GOS mimic HMOs are relatively unknown. Here we studied the effects of GOS utilization on a prominent gut symbiont, Bacteroides thetaiotaomicron, which has been previously shown to consume HMOs via mucin O-glycan degradation pathways. We show that several pathways for targeting O-mucin glycans are activated in B. thetaiotaomicron by GOS, as well as the galactan utilization sytem. Characterization of the endo-galactanase from this system identified activity on various longer GOS substrates while a subset of GOS compounds were identified as potential activators of mucin glycan metabolism in B. thetaiotaomicron. Our results show that GOS functions as an inducer of mucin-glycan pathways while providing a nutrient source in the form of ß-(1 → 4)-galactan. These metabolic features of GOS mixtures may serve to explain the beneficial effects that are seen for GOS supplemented infant formula.

Carbohydrate-binding module 74 is a novel starch-binding domain associated with large and multidomain α-amylase enzymes.

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore, we propose that this novel starch-binding domain is a new carbohydrate-binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence-based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut-associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract.

Structural and Functional Characterization of a Novel Family GH115 4-O-Methyl-α-Glucuronidase with Specificity for Decorated Arabinogalactans.

Glycoside hydrolases are clustered into families based on amino acid sequence similarities, and belonging to a particular family can infer biological activity of an enzyme. Family GH115 contains α-glucuronidases where several members have been shown to hydrolyze terminal α-1,2-linked glucuronic acid and 4-O-methylated glucuronic acid from the plant cell wall polysaccharide glucuronoxylan. Other GH115 enzymes show no activity on glucuronoxylan, and therefore, it has been proposed that family GH115 may be a poly-specific family. In this study, we reveal that a putative periplasmic GH115 from the human gut symbiont Bacteroides thetaiotaomicron, BtGH115A, hydrolyzes terminal 4-O-methyl-glucuronic acid residues from decorated arabinogalactan isolated from acacia tree. The three-dimensional structure of BtGH115A reveals that BtGH115A has the same domain architecture as the other structurally characterized member of this family, BoAgu115A; however the position of the C-terminal module is altered with respect to each individual enzyme. Phylogenetic analysis of GH115 amino sequences divides the family into distinct clades that may distinguish different substrate specificities. Finally, we show that BtGH115A α-glucuronidase activity is necessary for the sequential digestion of branched galactans from acacia gum by a galactan-ß-1,3-galactosidase from family GH43; however, while B. thetaiotaomicron grows on larch wood arabinogalactan, the bacterium is not able to metabolize acacia gum arabinogalactan, suggesting that BtGH115A is involved in degradation of arabinogalactan fragments liberated by other microbial species in the gastrointestinal tract.

Differential Metabolism of Exopolysaccharides from Probiotic Lactobacilli by the Human Gut Symbiont Bacteroides thetaiotaomicron.

Probiotic microorganisms are ingested as food or supplements and impart positive health benefits to consumers. Previous studies have indicated that probiotics transiently reside in the gastrointestinal tract and, in addition to modulating commensal species diversity, increase the expression of genes for carbohydrate metabolism in resident commensal bacterial species. In this study, it is demonstrated that the human gut commensal species Bacteroides thetaiotaomicron efficiently metabolizes fructan exopolysaccharide (EPS) synthesized by probiotic Lactobacillus reuteri strain 121 while only partially degrading reuteran and isomalto/malto-polysaccharide (IMMP) α-glucan EPS polymers. B. thetaiotaomicron metabolized these EPS molecules via the activation of enzymes and transport systems encoded by dedicated polysaccharide utilization loci specific for ß-fructans and α-glucans. Reduced metabolism of reuteran and IMMP α-glucan EPS molecules may be due to reduced substrate binding by components of the starch utilization system (sus). This study reveals that microbial EPS substrates activate genes for carbohydrate metabolism in B. thetaiotaomicron and suggests that microbially derived carbohydrates provide a carbohydrate-rich reservoir for B. thetaiotaomicron nutrient acquisition in the gastrointestinal tract.

Identifying the catalytic acid/base in GH29 α-L-fucosidase subfamilies.

While the catalytic nucleophile in the configuration-retaining α-L-fucosidases from family GH29 is fully conserved with respect to sequence, there is no fully sequence-conserved acid/base residue candidate across the family. X-ray crystallographic studies and kinetic characterizations have allowed the identification of this residue in a few cases, and a recent combination of phylogenetic tree analyses with substrate specificity data has allowed the division of GH29 enzymes into two subfamilies, A and B, allowing the probable assignment of these residues. Here, we perform detailed kinetic and mechanistic characterizations of the corresponding alanine mutants and other candidates. Through comparison of kinetic parameters obtained for the hydrolysis of fucosyl substrates with activated leaving groups by these mutants with those of the corresponding wild-type enzymes, in conjunction with the demonstration of azide rescue, we largely confirm the acid/base residue predictions for the GH29 fucosidases from the two subfamilies.

The conformation and function of a multimodular glycogen-degrading pneumococcal virulence factor.

SpuA is a large multimodular cell wall-attached enzyme involved in the degradation of glycogen by the pathogenic bacterium Streptococcus pneumoniae. The deletion of the gene encoding SpuA from the bacterium resulted in a strain with reduced competitiveness in a mouse model of virulence relative to the parent strain, linking the degradation of host-glycogen to the virulence of the bacterium. Through the combined use of X-ray crystallography, small-angle X-ray scattering, and inhibitor binding, the molecular features involved in substrate recognition by this complex protein are revealed. This uniquely illustrates the complexity of the active site, the conformational changes incurred during carbohydrate binding by this protein, and the interaction and cooperation of its composite modules during this process. New insight into the function of this particular pneumococcal virulence factor is provided along with substantial contributions to the nascent framework for understanding the structural and functional interplay between modules in multimodular carbohydrate-active enzymes.

The molecular basis of glycogen breakdown and transport in Streptococcus pneumoniae.

The genome of Streptococcus pneumoniae strains, as typified by the TIGR4 strain, contain several genes encoding proteins putatively involved in alpha-glucan degradation, modification and synthesis. The extracellular components comprise an ATP binding cassette-transporter with its solute binding protein, MalX, and the hydrolytic enzyme SpuA. We show that of the commonly occurring exogenous alpha-glucans, S. pneumoniae TIGR4 is only able to grow on glycogen in a MalX- and SpuA-dependent manner. SpuA is able to degrade glycogen into a ladder of alpha-1,4-glucooligosaccharides while the high-affinity interaction (K(a) approximately 10(6) M(-1)) of MalX with maltooligosaccharides plays a key role in promoting the selective uptake of the glycogen degradation products that are produced by SpuA. The X-ray crystallographic analyses of apo- and complexed MalX illuminate the protein's specificity for the degradation products of glycogen and its striking ability to recognize the helical structure of the ligand. Overall, the results of this work provide new structural and functional insight into streptococcal alpha-glucan metabolism while supplying biochemical support for the hypothesis that the substrate of the S. pneumoniaealpha-glucan metabolizing machinery is glycogen, which in a human host is abundant in lung epithelial cells, a common target for invasive S. pneumoniae.

Analysis of the reaction coordinate of alpha-L-fucosidases: a combined structural and quantum mechanical approach.

The enzymatic hydrolysis of alpha-L-fucosides is of importance in cancer, bacterial infections, and fucosidosis, a neurodegenerative lysosomal storage disorder. Here we show a series of snapshots along the reaction coordinate of a glycoside hydrolase family GH29 alpha-L-fucosidase unveiling a Michaelis (ES) complex in a (1)C(4) (chair) conformation and a covalent glycosyl-enzyme intermediate in (3)S(1) (skew-boat). First principles metadynamics simulations on isolated alpha-L-fucose strongly support a (1)C(4)<-->(3)H(4)<-->(3)S(1) conformational itinerary for the glycosylation step of the reaction mechanism and indicate a strong "preactivation" of the (1)C(4) complex to nucleophilic attack as reflected by free energy, C1-O1/O5-C1 bond length elongation/reduction, C1-O1 bond orientation, and positive charge development around the anomeric carbon. Analysis of an imino sugar inhibitor is consistent with tight binding of a chair-conformed charged species.

A structural and functional analysis of alpha-glucan recognition by family 25 and 26 carbohydrate-binding modules reveals a conserved mode of starch recognition.

Starch-hydrolyzing enzymes lacking alpha-glucan-specific carbohydrate-binding modules (CBMs) typically have lowered activity on granular starch relative to their counterparts with CBMs. Thus, consideration of starch recognition by CBMs is a key factor in understanding granular starch hydrolysis. To this end, we have dissected the modular structure of the maltohexaose-forming amylase from Bacillus halodurans (C-125). This five-module protein comprises an N-terminal family 13 catalytic module followed in order by two modules of unknown function, a family 26 CBM (BhCBM26), and a family 25 CBM (BhCBM25). Here we present a comprehensive structure-function analysis of starch and alpha-glucooligosaccharide recognition by BhCBM25 and BhCBM26 using UV methods, isothermal titration calorimetry, and x-ray crystallography. The results reveal that the two CBMs bind alpha-glucooligosaccharides, particularly those containing alpha-1,6 linkages, with different affinities but have similar abilities to bind granular starch. Notably, these CBMs appear to recognize the same binding sites in granular starch. The enhanced affinity of the tandem CBMs for granular starch is suggested to be the main biological advantage for this enzyme to contain two CBMs. Structural studies of the native and ligand-bound forms of BhCBM25 and BhCBM26 show a structurally conserved mode of ligand recognition but through non-sequence-conserved residues. Comparison of these CBM structures with other starch-specific CBM structures reveals a generally conserved mode of starch recognition.

Alpha-glucan recognition by a new family of carbohydrate-binding modules found primarily in bacterial pathogens.

TmPul13, a family 13 glycoside hydrolase from Thermotoga maritima, is a four-module protein having pullulanase activity; the three N-terminal modules are of unknown function while the large C-terminal module is likely the catalytic module. Dissection of the functions of the three unknown modules revealed that the 100 amino acid module at the extreme N-terminus of TmPul13 comprises a new family of carbohydrate-binding modules (CBM) that a bioinformatic analysis shows are most frequently found in pullulanase-like sequences from bacterial pathogens. Detailed binding studies of this isolated CBM, here called TmCBM41, reveals a preference for alpha-(1,4)-linked glucans, but occasional alpha-(1,6)-linked glucose residues, such as those found in pullulan, are tolerated. UV difference, isothermal titration calorimetry, and analytical ultracentrifugation binding studies suggest that maltooligosaccharides longer than four glucose residues are able to bind two TmCBM41 molecules per oligosaccharide when sugar concentrations are below the CBM concentration. This is explained in terms of an equilibrium expression involving the formation of both a 1 to 1 sugar to CBM complex and a 1 to 2 sugar to CBM complex (i.e., a CBM dimer ligated by an oligosaccharide). The presence of an alpha-(1-6) linkage in the oligosaccharide appears to prevent this phenomenon.

Binding sub-site dissection of a carbohydrate-binding module reveals the contribution of entropy to oligosaccharide recognition at "non-primary" binding subsites.

The optimal ligands for many carbohydrate-binding proteins are often oligosaccharides comprising two, three, or more monosaccharide units. The binding affinity for these sugars is increased incrementally by contributions from binding subsites on the protein that accommodate the individual monosaccharide residues of the oligosaccharide. Here, we use CsCBM6-1, a xylan-specific type B carbohydrate-binding module (CBM) from Clostridium stercorarium falling into amino acid sequence family CBM6, as a model system to investigate the structural and thermodynamic contributions of binding subsites in this protein to carbohydrate recognition. The three-dimensional structures of uncomplexed CsCBM6-1 (at 1.8 A resolution) and bound to the oligosaccharides xylobiose, xylotriose, and xylotetraose (at 1.70 A, 1.89 A, and 1.69 A resolution, respectively) revealed the sequential occupation of four subsites within the binding site in the order of subsites 2, 3, 4 then 1. Overall, binding to all of the xylooligosaccharides tested was enthalpically favourable and entropically unfavourable, like most protein-carbohydrate interactions, with the primary subsites 2 and 3 providing the bulk of the free energy and enthalpy of binding. In contrast, the contributions to the changes in entropy of the non-primary subsites 1 and 4 to xylotriose and xylotetraose binding, respectively, were positive. This observation is remarkable, in that it shows that the 10-20-fold improvement in association constants for oligosaccharides longer than a disaccharide is facilitated by favourable entropic contributions from the non-primary binding subsites.