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.

Structural Insight into a Yeast Maltase-The BaAG2 from Blastobotrys adeninivorans with Transglycosylating Activity.

An early-diverged yeast, Blastobotrys (Arxula) adeninivorans (Ba), has biotechnological potential due to nutritional versatility, temperature tolerance, and production of technologically applicable enzymes. We have biochemically characterized from the Ba type strain (CBS 8244) the GH13-family maltase BaAG2 with efficient transglycosylation activity on maltose. In the current study, transglycosylation of sucrose was studied in detail. The chemical entities of sucrose-derived oligosaccharides were determined using nuclear magnetic resonance. Several potentially prebiotic oligosaccharides with α-1,1, α-1,3, α-1,4, and α-1,6 linkages were disclosed among the products. Trisaccharides isomelezitose, erlose, and theanderose, and disaccharides maltulose and trehalulose were dominant transglycosylation products. To date no structure for yeast maltase has been determined. Structures of the BaAG2 with acarbose and glucose in the active center were solved at 2.12 and 2.13 Å resolution, respectively. BaAG2 exhibited a catalytic domain with a (ß/α)8-barrel fold and Asp216, Glu274, and Asp348 as the catalytic triad. The fairly wide active site cleft contained water channels mediating substrate hydrolysis. Next to the substrate-binding pocket an enlarged space for potential binding of transglycosylation acceptors was identified. The involvement of a Glu (Glu309) at subsite +2 and an Arg (Arg233) at subsite +3 in substrate binding was shown for the first time for α-glucosidases.

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.

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.

Identification and Characterization of a ß-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66T.

ß-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 ß-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66T, are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied ß-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc ß-1,4 and ß-1,3 linkages in chitobiose (GlcNAc)2 and GlcNAc-1,3-ß-Gal-1,4-ß-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: kcat/KM being 341 and 344 mM-1 s-1, respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide ß-Gal-1,4-ß-Glc-1,1-ß-GlcNAc, and in low amounts the ß-1,2- or ß-1,3-linked trisaccharide ß-Gal-1,4(ß-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 ß-N-acetylhexosaminidases.

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.

Structural characterization of bioactive heteropolysaccharides from the medicinal fungus Inonotus obliquus (Chaga).

The aim of this paper was to perform a comprehensive characterization of polysaccharides isolated from the interior (IOI) and exterior (IOE) parts of the fungus Inonotus obliquus. Pre-extraction with DCM and MeOH, followed by water and alkali extraction and ethanol precipitation gave two water extracts and two alkali extracts. Neutral and acidic polysaccharide fractions were obtained after anion-exchange chromatography of the water extracts. The neutral polysaccharides (60-73 kDa) were heterogeneous and branched and consisted of a (1 → 3)-linked ß-Glc backbone with (1 → 6)-linked kinks in the chain at approximately every fifth residue, with branches of (1 → 6)-linked ß-Glc in addition to substantial amounts of (1 → 6)-linked α-Gal with 3-O-methylation at about every third Gal residue. The acidic polysaccharide fractions (10-31 kDa) showed similar structural motifs as the neutral fractions differing mainly by the presence of (1 → 4)-linked α-GalA and α-GlcA. ß-Xyl, α-Man and α-Rha were also present in varying amounts in all fractions. No major structural differences between the IOI and IOE fractions were observed. An alkaline polysaccharide fraction (>450 kDa) was obtained from the IOI alkali extract, and consisted mainly of (1 → 3)- and (1 → 6)-linked ß-Glc and (1 → 4)-linked ß-Xyl. Several of the fractions showed in vitro immunomodulatory effect by increasing NO production in the murine macrophage and dendritic cell lines J774.A1 and D2SC/1. Most fractions managed to increase NO production only at the highest concentration tested (100 µg/ml), while the neutral fraction IOE-WN activated potent NO production at 10 µg/ml and was considered the most promising immunomodulating fraction in this study.