Promiscuous Yet Specific: A Methionine-Aromatic Interaction Drives the Reaction Scope of the Family 1 Glycosyltransferase GmUGT88E3 from Soybean.

Family 1 glycosyltransferases (GT1s, UGTs) catalyze the regioselective glycosylation of natural products in a single step. We identified GmUGT88E3 as a particularly promising biocatalyst able to produce a variety of pure, single glycosidic products from polyphenols with high chemical yields. We investigated this particularly desirable duality toward specificity, i.e., promiscuous toward acceptors while regiospecific. Using high-field NMR, kinetic characterization, molecular dynamics simulations, and mutagenesis studies, we uncovered that the main molecular determinant of GmUGT88E3 specificity is a methionine-aromatic bridge, an interaction often present in protein structures but never reported for enzyme-substrate interactions. Here, mutating Met127 led to inactive proteins or 100-fold reduced activity.

The Engineered Hexosaminidase TtOGA-D120N Is an Efficient O-/N-/S-Glycoligase That Also Catalyzes Formation and Release of Oxazoline Donors for Cascade Syntheses with Glycosynthases or Transglycosylases.

Engineering glycoside hydrolases is a major route to obtaining catalysts forming glycosidic bonds. Glycosynthases, thioglycoligases, and transglycosylases represent the main strategies, each having advantages and drawbacks. Here, we show that an engineered enzyme from the GH84 family, the acid-base mutant TtOGA-D120N, is an efficient O-, N-, and S-glycoligase, able to use Ssp3, Osp3, Nsp2, and Nsp nucleophiles. Moreover, TtOGA-D120N catalyzes the formation and release of N-acetyl-d-glucosamine 1,2-oxazoline, the intermediate of hexosaminidases displaying substrate-assisted catalysis. This release of an activated intermediate allows cascade synthesis by combination with transglycosylases or glycosynthases, here exemplified by synthesis of the human milk oligosaccharide lacto-N-triose II.

Family 1 Glycosyltransferase UGT706F8 from Zea mays Selectively Catalyzes the Synthesis of Silibinin 7-O-ß-d-Glucoside.

Regioselective glycosylation is a chemical challenge, leading to multistep syntheses with protecting group manipulations, ultimately resulting in poor atom economy and compromised sustainability. Enzymes allow eco-friendly and regioselective bond formation with fully deprotected substrates in a single reaction. For the selective glucosylation of silibinin, a pharmaceutical challenged with low solubility, enzyme engineering has previously been employed, but the resulting yields and k cat were limited, prohibiting the application of the engineered catalyst. Here, we identified a naturally regioselective silibinin glucosyltransferase, UGT706F8, a family 1 glycosyltransferase from Zea mays. It selectively and efficiently (k cat = 2.1 ± 0.1 s-1; K M = 36.9 ± 5.2 µM; TTN = 768 ± 22) catalyzes the quantitative synthesis of silibinin 7-O-ß-d-glucoside. We solved the crystal structure of UGT706F8 and investigated the molecular determinants of regioselective silibinin glucosylation. UGT706F8 was the only regioselective enzyme among 18 glycosyltransferases found to be active on silibinin. We found the temperature optimum of UGT706F8 to be 34 °C and the pH optimum to be 7-8. Our results indicate that UGT706F8 is an efficient silibinin glycosyltransferase that enables biocatalytic production of silbinin 7-O-ß-d-glucoside.

Full NMR assignment, revised structure and biosynthetic analysis for the capsular polysaccharide from Streptococcus Pneumoniae serotype 15F.

Upon investigation of Streptococcus pneumoniae serotype 15F capsular polysaccharide (CPS), we discovered that it had a different phosphorylation substituent, namely glycerol-2-phosphate like the other serogroup 15 CPS rather than the originally reported 0.2 equivalent of phosphate or phosphocholine. Furthermore, we also determined the locations of the two previously unassigned O-acetyl groups present in the repeating unit of the 15F CPS, and carried out full NMR assignments of the 15F as well as 15A CPS. Lastly, a biosynthetic analysis of serotypes 15F and 15A was performed and used to make a prediction for the structure of the recently discovered serotype 15D.

Binding Sites for Oligosaccharide Repeats from Lactic Acid Bacteria Exopolysaccharides on Bovine ß-Lactoglobulin Identified by NMR Spectroscopy.

Lactic acid bacterial exopolysaccharides (EPS) are used in the food industry to improve the stability and rheological properties of fermented dairy products. ß-Lactoglobulin (BLG), the dominant whey protein in bovine milk, is well known to bind small molecules such as fatty acids, vitamins, and flavors, and to interact with neutral and anionic polysaccharides used in food and pharmaceuticals. While sparse data are available on the affinity of EPS-milk protein interactions, structural information on BLG-EPS complexes, including the EPS binding sites, is completely lacking. Here, binding sites on BLG variant A (BLGA), for oligosaccharides prepared by mild acid hydrolysis of two EPS produced by Streptococcus thermophilus LY03 and Lactobacillus delbrueckii ssp. bulgaricus CNRZ 1187, respectively, are identified by NMR spectroscopy and supplemented by isothermal titration calorimetry (ITC) and molecular docking of complexes. Evidence of two binding sites (site 1 and site 2) on the surface of BLGA is achieved for both oligosaccharides (LY03-OS and 1187-OS) through NMR chemical shift perturbations, revealing multivalency of BLGA for EPS. The affinities of LY03-OS and 1187-OS for BLGA gave K D values in the mM range obtained by both NMR (pH 2.65) and ITC (pH 4.0). Molecular docking suggested that the BLGA and EPS complexes depend on hydrogen bonds and hydrophobic interactions. The findings provide insights into how BLGA engages structurally different EPS-derived oligosaccharides, which may facilitate the design of BLG-EPS complexation, of relevance for formulation of dairy products and improve understanding of BLGA coacervation.

Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases.

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6-12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d/l-, α/ß-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.

Structural, Genetic, and Serological Elucidation of Streptococcus pneumoniae Serogroup 24 Serotypes: Discovery of a New Serotype, 24C, with a Variable Capsule Structure.

Pneumococcal capsules are important in pneumococcal pathogenesis and vaccine development. Although conjugate vaccines have brought about a significant reduction in invasive pneumococcal disease (IPD) caused by vaccine serotypes, the relative serotype prevalence has shifted with the dramatic emergence of serotype 24F in some countries. Here, we describe 14 isolates (13 IPD and 1 non-IPD) expressing a new capsule type, 24C, which resembles 24F but has a novel serological profile. We also describe the antigenic, biochemical, and genetic basis of 24F and 24C and the related serotypes 24A and 24B. Structural studies show that 24B, 24C, and 24F have identical polysaccharide backbones [ß-Ribf-(1→4)-α-Rhap-(1→3)-ß-GlcpNAc-(1→4)-ß-Rhap-(1→4)-ß-Glcp] but with different side chains, as follows: 24F has arabinitol-phosphate and 24B has ribitol-phosphate. 24C has a mixture of 24F and 24B repeating units, with the ratio of ribitol to arabinitol being strain dependent. In contrast, the 24A capsule has a backbone without ß-Ribf but with arabinitol-phosphate and phosphocholine side chains. These structures indicate that factor-sera 24d and 24e recognize arabinitol and ribitol, respectively, which explains the serology of serogroup 24, including those of 24C. The structures can be genetically described by the bispecificity of wcxG, which is capable of transferring arabinitol or ribitol when arabinitol is limiting. Arabinitol is likely not produced in 24B but is produced in reduced amounts in 24C due to various mutations in abpA or abpB genes. Our findings demonstrate how pneumococci modulate their capsule structure and immunologic properties with small genetic changes, thereby evading host immune responses. Our findings also suggest a potential for new capsule types within serogroup 24.

Structural, biosynthetic and serological cross-reactive elucidation of capsular polysaccharides from Streptococcus pneumoniae serogroup 28.

Capsular polysaccharides (CPS) are the key virulent factors in the pathogenesis of Streptococcus pneumoniae. The previously unknown CPS structures of the pneumococcal serotype 28F and 28A were thoroughly characterized by NMR spectroscopy, chemical analysis and AF4-MALS-dRI. The following repeat unit structures were determined: -4)[α-l-Rhap-[4-P-2-Gro]]-(1-3)-α-d-Sug-[6-P-Cho]-(1-3)-ß-l-Rhap-[2-OAc]-(1-4)-ß-d-Glcp-(1-; 28F: Sug = Glcp, Mw: 540.5 kDa; 28A: Sug = GlcpNAc, Mw: 421.9 kDa; The correlation of CPS structures with biosynthesis showed that glycosyltransferase WciU in serotypes 28F and 28A had different sugar donor specificity toward α-d-Glcp and α-d-GlcNAcp, respectively. Furthermore, latex agglutination tests of de-OAc and de-PO4 CPS were conducted to understand cross-reactions between serogroup 28 with factor antiserum 23d. Interestingly, the de-OAc 28F and 28A CPS can still weakly react with factor antiserum 23d, while de-PO4 CPS did not react with factor antiserum 23d. This indicated that OAc group could affect the affinity and P-2-Gro was crucial for cross-reacting with factor antiserum 23d.

Unexpected Anomeric Acceptor Preference Observed Using dDNP NMR for Transglycosylation Studies of ß-Galactosidases.

The transglycosylation abilities of ß-galactosidases were investigated using hyperpolarized [U-13C,U-2H]glucose as an acceptor and o-nitrophenyl ß-galactopyranoside as a donor. Several products were readily observable, and at least in the case when O3 acted as an acceptor, the enzymes showed a clear selectivity toward the ß-anomer of glucose. Additionally, it was possible to determine the relative hydrolysis rates of the formed transglycosylation products, providing information on the selectivity as well. Using this method, the transglycosylation abilities of the enzymes could be studied at a very high temporal resolution as well as with high sensitivity, and due to the relative ease of the setup, this method could be more generally applied to investigate glycosidases.

Structural, Biosynthetic, and Serological Cross-Reactive Elucidation of Capsular Polysaccharides from Streptococcus pneumoniae Serogroup 16.

Capsular polysaccharides (CPS) are crucial virulence factors of Streptococcus pneumoniae The previously unknown CPS structures of the pneumococcal serogroup 16 (serotypes 16F and 16A) were thoroughly elucidated by nuclear magnetic resonance (NMR) spectroscopy and verified by chemical analysis. The following repeat unit structures were determined: 16F, -3)-α-l-Rhap-[4-P-1-Gro]-(1-3)-α-d-Glcp-[(6-P-1)-Gro]-(1-3)-ß-l-Rhap-[2-OAc]-(1-4)-ß-d-Glcp-(1-; 16A, -3)-ß-d-Galf-[2-OAc (70%)]-(1-3)-α-l-Rhap-(1-2)-α-l-Rhap-(1-3)-α-d-Galp-[(6-P-1)-Gro]-(1-3)-ß-d-Galp-(1-4)-ß-d-Glcp-(1- (OAc, O-acetyl substitution; P-1-Gro, glycerol-1-phosphate substitution) A further analysis of CPS biosynthesis of serotypes 16F and 16A, in conjunction with published cps gene bioinformatics analysis and structures of related serotypes, revealed presumable specific function of glycosyltransferase, acetyltransferase, phosphotransferase, and polymerase. The functions of glycosyltransferases WcxN and WcxT were proposed for the first time, and they were assigned to catalyze linkage of α-l-Rhap-(1-3)-α-d-Glcp and α-l-Rhap-(1-2)-α-l-Rhap, respectively. Furthermore, since serotype 16F was genetically close to serogroup 28, cross-reactions between serogroup 16 and serogroup 28 were studied using diagnostic antisera, which provided further understanding of antigenic properties of CPS and diagnostic antisera. Interestingly, serotype 16F cross-reacted with factor antisera 28b and 11c. Meanwhile, serotype 16A cross-reacted with factor antiserum 11c.IMPORTANCE The vaccine pressure against Streptococcus pneumoniae could result in a change of prevalence in carriage and invasive serotypes. As such, it is necessary to monitor the distribution to achieve successful vaccination of the population, and similarly, it is important to increase the knowledge of even the currently less prevalent serotypes. The CPS are vital for the virulence of the pathogen, and antigenic properties of CPS are based on the structure. Consequently, a better understanding of the structure, biosynthesis, and serology of the capsular polysaccharides can be of great importance toward developing future diagnostic tools and vaccines.

Alginate Trisaccharide Binding Sites on the Surface of ß-Lactoglobulin Identified by NMR Spectroscopy: Implications for Molecular Network Formation.

ß-lactoglobulin (BLG) is a promiscuous protein in terms of ligand interactions, having several binding sites reported for hydrophobic biomolecules such as fatty acids, lipids, and vitamins as well as detergents. BLG also interacts with neutral and anionic oligo- and polysaccharides for which the binding sites remain to be identified. The multivalency offered by these carbohydrate ligands is expected to facilitate coacervation, an electrostatically driven liquid-liquid phase separation. Using heteronuclear single quantum coherence NMR spectroscopy and monitoring chemical shift perturbations, we observed specific binding sites of modest affinity for alginate oligosaccharides (AOSs) prepared by alginate lyase degradation. Two different AOS binding sites (site 1 and site 2) centered around K75 and K101 were identified for monomeric BLG isoform A (BLGA) at pH 2.65. In contrast, only site 1 around K75 was observed for dimeric BLGA at pH 4.0. The data suggest a pH-dependent mechanism whereby both the BLGA dimer-monomer equilibrium and electrostatic interactions are exploited. This variability allows for control of coacervation and particle formation of BLGA/alginate mixtures via directed polysaccharide bridging of AOS binding sites and has implication for molecular network formation. The results are valuable for design of polyelectrolyte-based BLG particles and coacervates for carrying nutraceuticals and modulating viscosity in dairy products by use of alginates.

Discovery and description of a new serogroup 7 Streptococcus pneumoniae serotype, 7D, and structural analysis of 7C and 7D.

Streptococcus pneumoniae is characterised into 92 serotypes based on antigenic reactions of commercial rabbit sera to the capsular polysaccharides. During development of a bioinformatic serotyping tool (PneumoCaT), an isolate exhibited a novel codon at residue 385 of the glycosyltransferase gene wcwK encoding a distinct amino acid, which differentiates genogroup 7. Investigation by repeat serotyping and Quellung reaction revealed a novel pattern of factor sera with the isolate reacting very strongly with 7f, but also with 7e factor sera. The structure of the capsular polysaccharide was determined by NMR spectroscopy to be an approximately 5:1 combination of the structures of 7C and 7B, respectively, and the structure of 7C was also elucidated. All data from whole genome sequencing, NMR spectroscopy, production of antisera and serotyping of the novel 7 strain shows that it is a new serotype, which will be named in the Danish nomenclature as 7D.

Discovery of Intermediates of lacZ ß-Galactosidase Catalyzed Hydrolysis Using dDNP NMR.

Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state 13C NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ ß-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction intermediates were observed, including a 1,1-linked disaccharide. The enzyme is known for making 1,6-transglycosylation, producing products like allolactose, that are also substrates. To analyze the kinetics, a simple kinetic model was developed and used to determine relative transglycosylation and hydrolysis rates of each of the intermediates, and the novel transglycosylation intermediates were determined as better substrates than the 1,6-linked one, explaining their transient nature. These findings suggest that hydrolysis and transglycosylation might be more complex than previously described.

A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galß1-4GlcNAc) and LacdiNAc (GalNAcß1-4GlcNAc) motifs found on vertebrate and insect cells.

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galß1-4GlcNAcß-tetramethylrhodamine (LacNAc-TMR (Galß1-4GlcNAcß(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcß1-4GlcNAcß-TMR), LacNAc-TMR, and LacNAcß1-6LacNAcß-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the ß1-6 bond in LacNAcß1-6LacNAcß-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.

In vitro growth of four individual human gut bacteria on oligosaccharides produced by chemoenzymatic synthesis.

The present study aimed at examining oligosaccharides (OS) for potential stimulation of probiotic bacteria. Nineteen structurally well-defined candidate OS covering groups of ß-glucosides, α-glucosides and α-galactosides with degree of polymerization 2-4 were prepared in >100 mg amounts by chemoenzymatic synthesis (i.e. reverse phosphorolysis or transglycosylation). Fourteen of the OS are not naturally occurring and five (ß-D-glucosyl-fructose, ß-D-glucosyl-xylitol, α-glucosyl-(1,4)-D-mannose, α-glucosyl-(1,4)-D-xylose; α-glucosyl-(1,4)-L-fucose) have recently been synthesized for the first time. These OS have not been previously tested for effects of bacterial growth and here the ability of all 19 OS to support growth of four gastrointestinal bacteria: three probiotic bacteria Bifidobacterium lactis, Bifidobacterium longum, and Lactobacillus acidophilus, and one commensal bacterium, Bacteroides vulgatus has been evaluated in monocultures. The disaccharides ß-D-glucosyl-xylitol and ß-D-glucosyl-(1,4)-xylose noticeably stimulated growth yields of L. acidophilus NCFM, and additionally, ß-D-glucosyl-(1,4)-xylose stimulated B. longum Bl-05. α-Glucosyl-(1,4)-glucosamine and α-glucosyl-(1,4)-N-acetyl-glucosamine enhanced the growth rate of B. animalis subsp. lactis and B. longum Bl-05, whereas L. acidophilus NCFM and Bac. vulgatus did not grow on these OS. α-Galactosyl-(1,6)-α-galactosyl-(1,6)-glucose advanced the growth rate of B. animalis subsp. lactis and L. acidophilus NCFM. Thus several of the structurally well-defined OS supported growth of beneficial gut bacteria. This reflects a broad specificity of their sugar transporters for OS, including specificity for non-naturally occurring OS, hence showing promise for design of novel prebiotics.

NMR assignment of structural motifs in intact ß-limit dextrin and its α-amylase degradation products in situ.

An increasingly detailed and realistic view of biological processes often hinges on atomic-level characterization of biomacromolecules and of the processes they are involved in, preferably under near-physiological conditions. Structure, degradation, and synthesis of glucose storage polymers have been studied for decades with a range of analytical tools, but the detailed in situ analysis has remained an analytical challenge. Here, we report the NMR assignment of different structural motifs in the ß-limit dextrin from lintnerized maize starch as a branched α-glucan model system for starch, which is depleted of repetitive α-(1→4) glycosidic bonds at non-reducing ends but has the α-(1→6) branch points intact. By NMR spectroscopy at 18.7T magnetic field, we assign 12 discernible α-glucopyranosyl spin systems and identify them with different structural motifs. Amylolysis of the ß-limit dextrin is directly followed by real-time NMR spectroscopy and four major cleavage products are identified and assigned to different branch point structures. Overall, these NMR assignments facilitate in situ assays under realistic conditions of substrate competition, transglycosylation, and product inhibition and shed light on chemical shift tendencies in different structural motifs of branched α-glucans.

Atlantinone A, a Meroterpenoid Produced by Penicillium ribeum and Several Cheese Associated Penicillium Species.

Atlantinone A has been isolated from the psychrotolerant fungus Penicillium ribeum. The exact structure of the compound was confirmed by mass spectrometric and 1- and 2D NMR experiments. Atlantinone A was originally only produced upon chemical epigenetic manipulation of P. hirayamae, however in this study the compound was found to be produced at standard growth conditions by the following species; P. solitum, P. discolor, P. commune, P. caseifulvum, P. palitans, P. novae-zeelandiae and P. monticola. A biosynthetic pathway to atlantinone A starting from andrastin A is proposed.

Chemical and biological characterization of pectin-like polysaccharides from the bark of the Malian medicinal tree Cola cordifolia.

The bark of Cola cordifolia used in Malian traditional medicine contains unusual types of polysaccharides with immunomodulating activities. We report for the first time on the structure of a polymer designated CC1P1 having the repeating structure [2→)[α-D-Gal(1→3)]α-L-Rha(1→4)α-d-GalA(1→] as determined by NMR and GC/MS. α-Linked Gal is unusual in pectins. The Mw of 135 kDa was determined by SEC-MALLS. CC1P2 (1400 kDa), another polymer, having the same backbone, but this was substituted with α-4-OMe-GlcA, α-2-OMe-Gal and α-Gal as terminal units. CC1P1 shows a high complement-fixing activity, IC50 being 2.2 times lower than the positive pectin control PMII (IC50 appr. 71 µg/mL) while IC50 of CC1P2 is 1.8 times lower. The simple structure of CC1P1 did not activate macrophages, while CC1P2 (100 µg/mL) showed the same potency as the positive controls PMII (100 µg/mL) and LPS (500 ng/mL). No cytotoxicity was detected.

Real-time detection of central carbon metabolism in living Escherichia coli and its response to perturbations.

The direct tracking of cellular reactions in vivo has been facilitated with recent technologies that strongly enhance NMR signals in substrates of interest. This methodology can be used to assay intracellular reactions that occur within seconds to few minutes, as the NMR signal enhancement typically fades on this time scale. Here, we show that the enhancement of (13)C nuclear spin polarization in deuterated glucose allows to directly follow the flux of glucose signal through rather extended reaction networks of central carbon metabolism in living Escherichia coli. Alterations in central carbon metabolism depending on the growth phase or upon chemical perturbations are visualized with minimal data processing by instantaneous observation of cellular reactions.