Investigation into the efficiency of diverse N-linking oligosaccharyltransferases for glycoengineering using a standardised cell-free assay.

The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity.

Endo- and exo-levanases from Bacillus subtilis HM7: Catalytic components, synergistic cooperation, and application in fructooligosaccharide synthesis.

Levan-type fructooligosaccharides (LFOS) exhibit significant biological activities and selectively promote the growth of certain beneficial bacteria. Levanase is an important enzyme for LFOS production. In this study, two isoforms of levanases, exo- and endo-type depolymerizing enzymes, from Bacillus subtilis HM7 isolated from Dynastes hercules larvae excrement were cloned, expressed, and characterized. The synergistic effect on the levan hydrolysis and kinetic properties of both isoforms were evaluated, indicating their cooperation in levan metabolism, where the endo-levanase catalyzes a rate-limiting step. In addition, homology models and molecular dynamics simulations revealed the key amino residues of the enzymes for levan binding and catalysis. It was found that both isoforms possessed distinct binding residues in the active sites, suggesting the importance of the specificity of the enzymes. Finally, we demonstrated the potential of endo-type levanase in LFOS synthesis using a one-pot reaction with levansucrase. Overall, this study fills the knowledge gap in understanding levanase's mechanism, making an important contribution to the fields of food science and biotechnology.

Characterization of a novel inulosucrase from Lactiplantibacillus plantarum.

Fructansucrases produce fructans by polymerizing the fructose moiety released from sucrose. Here, we describe the recombinant expression and characterization of a unique fructansucrase from Lactiplantibacillus plantarum DKL3 that showed low sequence similarity with previously characterized fructansucrases. The optimum pH and temperature of fructansucrase were found to be 4.0 and 35 °C, respectively. Enzyme activity increased in presence of Ca2+ and distinctly in presence of Mn2+. The enzyme was characterized as an inulosucrase (LpInu), based on the production of an inulin-type fructan as assessed byNMR spectroscopy and methylation analysis. In addition to ß-2,1-linkages, the inulin contained a few ß-2,1,6-linked branchpoints. High-performance size exclusion chromatography with refractive index detection (HPSEC-RI) revealed the production of inulin with a lower molecular weight compared to other characterized bacterial inulin. LpInu and its inulin product represent novel candidates to be explored for possible food and biomedical applications.

Production of prebiotic enriched maple syrup through enzymatic conversion of sucrose into fructo-oligosaccharides.

Maple syrup, a popular natural sweetener has a high content of sucrose, whose consumption is linked to different health issues such as obesity and diabetes. Hence, within this paper, the conversion of sucrose to prebiotics (fructo-oligosaccharides, FOS) was proposed as a promising approach to obtaining a healthier, value-added product. Enzymatic conversion was optimized with respect to key experimental factors, and thereafter derived immobilized preparation of fructosyltransferase (FTase) from Pectinex® Ultra SP-L (FTase-epoxy Purolite, 255 IU/g support) was successfully utilized to produce novel functional product in ten consecutive reaction cycles. The product, obtained under optimal conditions (60 °C, 7.65 IU/mL, 12 h), resulted in 56.0% FOS, 16.7% sucrose, and 27.3% monosaccharides of total carbohydrates, leading to a 1.6-fold reduction in caloric content. The obtained products` prebiotic potential toward the probiotic strain Lactobacillus plantarum 299v was demonstrated. The changes in physico-chemical and sensorial characteristics were esteemed as negligible.

Investigating the Potential of Phenolic Compounds and Carbohydrates as Acceptor Substrates for Levansucrase-Catalyzed Transfructosylation Reaction.

This study characterizes the acceptor specificity of levansucrases (LSs) from Gluconobacter oxydans (LS1), Vibrio natriegens (LS2), Novosphingobium aromaticivorans (LS3), and Paraburkholderia graminis (LS4) using sucrose as fructosyl donor and selected phenolic compounds and carbohydrates as acceptors. Overall, V. natriegens LS2 proved to be the best biocatalyst for the transfructosylation of phenolic compounds. More than one fructosyl unit could be attached to fructosylated phenolic compounds. The transfructosylation of epicatechin by P. graminis LS4 resulted in the most diversified products, with up to five fructosyl units transferred. In addition to the LS source, the acceptor specificity of LS towards phenolic compounds and their transfructosylation products were found to greatly depend on their chemical structure: the number of phenolic rings, the reactivity of hydroxyl groups and the presence of aliphatic chains or methoxy groups. Similarly, for carbohydrates, the transfructosylation yield was dependent on both the LS source and the acceptor type. The highest yield of fructosylated-trisaccharides was Erlose from the transfructosylation of maltose catalyzed by LS2, with production reaching 200 g/L. LS2 was more selective towards the transfructosylation of phenolic compounds and carbohydrates, while reactions catalyzed by LS1, LS3 and LS4 also produced fructooligosaccharides. This study shows the high potential for the application of LSs in the glycosylation of phenolic compounds and carbohydrates.

Reconstitution and resonance assignments of yeast OST subunit Ost4 and its critical mutant Ost4V23D in liposomes by solid-state NMR.

N-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in N-linked glycosylation pathways result in metabolic diseases collectively called Congenital Disorders of Glycosylation. In this modification reaction, a mannose rich oligosaccharide is transferred from a lipid-linked donor substrate to a specific asparagine side-chain within the -N-X-T/S- sequence (where X ≠ Proline) of the nascent protein. Oligosaccharyltransferase (OST), a multi-subunit membrane embedded enzyme catalyzes this glycosylation reaction in eukaryotes. In yeast, Ost4 is the smallest of nine subunits and bridges the interaction of the catalytic subunit, Stt3, with Ost3 (or its homolog, Ost6). Mutations of any C-terminal hydrophobic residues in Ost4 to a charged residue destabilizes the enzyme and negatively impacts its function. Specifically, the V23D mutation results in a temperature-sensitive phenotype in yeast. Here, we report the reconstitution of both purified recombinant Ost4 and Ost4V23D each in a POPC/POPE lipid bilayer and their resonance assignments using heteronuclear 2D and 3D solid-state NMR with magic-angle spinning. The chemical shifts of Ost4 changed significantly upon the V23D mutation, suggesting a dramatic change in its chemical environment.

Innovative application of a novel and thermostable inulin fructotransferase from Arthrobacter sp. ISL-85 to fructan inulin in burdock root to improve nutrition.

Inulin fructotransferase converts prebiotic polysaccharide inulin to difructose anhydride III, known for its numerous beneficial physiological effects. While previous studies focused on using inulin extracts under optimal conditions, this study delves into the enzyme's behavior when dealing with more complex food materials, inulin-rich burdock root, which possesses greater nutritional value but may influence the enzymatic reaction. An inulin fructotransferase from Arthrobacter sp. ISL-85 was identified and characterized, which has the highest activity of 783 U mg-1 at pH 6.5 and 65 °C and remains stable even up to 80 °C. When applied to inulin-rich burdock root (pH 4.7) at 80 °C for 2 h, the enzyme yielded 4.1 g of difructose anhydride III, concurrently increasing fructo-oligosaccharides. This study demonstrates the potential of this enzyme as a valuable tool for efficiently processing inulin within whole food materials under high temperatures. Such an approach could pave the way for enhancing nutrition and promoting health benefits.

Similarities and differences of nano-sized levan synthesized by Bacillus haynesii at low and high temperatures: Characterization and bioactivity.

Levan is a biopolymer with many different uses. Temperature is an important parameter in biopolymer synthesis. Herein, levan production was carried out from Bacillus haynesii, a thermophilic microorganism, in the temperature range of 4 °C-95 °C. The highest levan production was measured as 10.9 g/L at 37 °C. The synthesized samples were characterized by FTIR and NMR analysis. The particle size of the levan samples varied between 153 and 824.4 nm at different temperatures. In levan samples produced at high temperatures, the water absorption capacity is higher in accordance with the particle size. Irregularities were observed in the surface pores at temperatures of 60 °C and above. The highest emulsion capacity of 83.4 % was measured in the sample synthesized at 4 °C. The antioxidant activity of all levan samples synthesized at different temperatures was measured as 84 % on average. All synthesized levan samples showed antibacterial effect on pathogenic bacteria. In addition, levan synthesized at 45 °C showed the highest antimicrobial effect on E. coli ATCC 35218 with an inhibition zone of 21.3 ± 1.82 mm. Antimicrobial activity against yeast sample C. albicans, was measured only in levan samples synthesized at 80 °C, 90 °C, 95 °C temperatures. Levan synthesized from Bacillus haynesii at low and high temperatures showed differences in characterization and bioactivity.

Rational design of a self-assembly promoting fusion domain enhances high molecular weight levan synthesis by levansucrase SacB.

The catalytic product of levansucrase from Bacillus subtilis (SacB) is mainly composed of 10 % high molecular weight levan (HMW, ~2000 kDa) and 90 % low molecular weight levan (LMW, ~7000 Da). In order to achieve efficient production of food hydrocolloid, high molecular weight levan (HMW), with the help of molecular dynamics simulation software, a protein self-assembly element, Dex-GBD, was found and fused with the C-terminus of SacB to construct a novel fusion enzyme, SacB-GBD. The product distribution of SacB-GBD was reversed compared with SacB, and the proportion of HMW in the total polysaccharide was significantly increased to >95 %. We then confirmed that the self-assembly was responsible for the reversal of the SacB-GBD product distribution by the simultaneous modulation of SacB-GBD particle size and product distribution by SDS. The hydrophobic effect may be the main driver of self-assembly as analyzed by molecular simulations and hydrophobicity determination. Our study provides an enzyme source for the industrial production of HMW and provides a new theoretical basis for guiding the molecular modification of levansucrase towards the size of the catalytic product.

Chemo-Enzymatic Synthesis of Non-ribosomal Macrolactams by a Penicillin-Binding Protein-Type Thioesterase.

Penicillin-binding protein-type thioesterases (PBP-type TEs) are an emerging family of non-ribosomal peptide cyclases. PBP-type TEs exhibit distinct substrate scopes from the well-exploited ribosomal peptide cyclases and traditional non-ribosomal peptide cyclases. Their unique properties, as well as their stand-alone nature, highlight PBP-type TEs as valuable candidates for development as biocatalysts for peptide macrocyclization. Here in this chapter, we describe the scheme for the chemoenzymatic synthesis of non-ribosomal macrolactam by SurE, a representative member of PBP-type TEs.

Successful Manipulation of the Product Spectrum of the Erwinia amylovora Levansucrase by Modifying the Residues around loop1, Loop 3, and Loop 4.

Levansucrase (LS, EC 2.4.1.10) catalyzes the synthesis of levan by successively transferring the fructosyl moiety from sucrose to an elongated fructan chain. Although the product distribution of LS from Erwinia amylovora (Ea-LS) was studied under different sucrose concentrations, the effect of residues on the product formation is yet unknown. The first levanhexaose-complexed structure of LS from Bacillus subtilis (Bs-SacB) provided information on the oligosaccharide binding sites (OB sites), from +1 to +4 subsites. Since Ea-LS would efficiently produce fructooligosaccharides, a substitution mutation of OB sites in Bs-SacB and the corresponding residues of Ea-LS were conducted to investigate how these mutants would influence the product distribution. As a result, a series of mutants with different product spectrum were obtained. Notably, the mutants of G98E, V151F, and N200T around loop 1, loop 3, and loop 4 all showed a significant increase in both the molecular mass and the yield of high-molecular-mass levan, suggesting that the product profile of Ea-LS was significantly modified.

Production and bioactivities of nanoparticulated and ultrasonic-degraded levan generated by Erwinia tasmaniensis levansucrase in human osteosarcoma cells.

Levan is a bioactive polysaccharide that can be synthesized by various microorganisms. In this study, the physicochemical properties and bioactivity of levan synthesized by recombinant levansucrase from Erwinia tasmaniensis were investigated. The synthesis conditions, including the enzyme concentration, substrate concentration, and temperature, were optimized. The obtained levan generally appeared as a cloudy suspension. However, it could transform into a hydrogel at concentrations exceeding 10 % (w/v). Then, ultrasonication was utilized to reduce the molecular weight and increase the bioavailability of levan. Dynamic light scattering (DLS) and gel permeation chromatography (GPC) indicated that the size of levan was significantly decreased by ultrasonication, whereas Fourier transform infrared spectroscopy, 1H-nuclear magnetic resonance, and X-ray powder diffraction revealed that the chemical structure of levan was not changed. Finally, the bioactivities of both levan forms were examined using human osteosarcoma (Saos-2) cells. The result clearly illustrated that sonicated levan had higher antiproliferative activity in Saos-2 cells than original levan. Sonicated levan also activated Toll-like receptor expression at the mRNA level. These findings suggested the important beneficial applications of sonicated levan for the development of cancer therapies.

Production of Human ABC Transporters and Oligosaccharyltransferase Complexes for Structural Studies.

Structural studies of membrane proteins require high-quality samples. The target proteins should not only be pure and homogeneous but should also be active and allow the capture of a functionally relevant state. Here we present optimized methods for the expression and purification of human ABC transporters and oligosaccharyltransferase (OST) complexes that can be used for high-resolution structure determination using single-particle cryo-electron microscopy (cryo-EM). The protocols are based on the generation of stable cell lines that enable tetracycline-inducible expression of the target proteins. For the multidrug exporter ABCB1, we describe a protocol for reconstitution into nanodiscs and evaluation of the ATPase activity in the presence of drugs. For human OST, we describe a strategy for the purification of OST-A and OST-B complexes, including techniques to evaluate their integrity and activity using in vitro glycosylation assays. These protocols can be adapted for the production of other human ABC transporters and multimeric membrane protein complexes.

Complete secretion of recombinant Bacillus subtilis levansucrase in Pichia pastoris for production of high molecular weight levan.

Three signal peptides from α-mating factor (α-MF), inulinase (INU) and native levansucrase (LS) were compared for secretion efficiency of Bacillus subtilis levansucrase SacB-T305A in Pichia pastoris GS115. The first complete secretion of bacterial levansucrase in yeasts under methanol induction was achieved while using α-MF signal. The secreted recombinant Lev(α-MF) proved to be glycosylated by combination of NanoLC-MS/MS and Endo H digestion. Interestingly, glycosylation not only improved significantly the polymerase thermostability, but also reversed the products profiles to favor synthesis of high molecular weight (HMW) levan which accounted for approximately 73 % to total levan-type polysaccharides. It indicated for the first time that the glycosylation of recombinant B. subtilis levansucrase affected significantly the products molecular weight distribution. It also provided a promising enzymatic way to effectively product HMW levan from sucrose resources.

Structural characterization of levan synthesized by a recombinant levansucrase and its application as yogurt stabilizers.

Levan, a class of fructan with complex structure, has been found to have potential as a prebiotic. In this study, the polysaccharide was produced by in vitro catalysis (Cata-lev), structural analysis proved it was levan that possessed a branching degree of about 15% and molecular weight of 2.54 × 106 Da. Furthermore, SEM observation showed that Cata-lev was porous with loose flaky structure. Subsequently, the effects of five Cata-lev concentrations on the fermentation process, texture, nutraceutical, and sensory characteristics of set yogurts were investigated and compared to Fructooligosaccharides (FOS). The addition of Cata-lev could significantly (P < 0.05) increase the water holding capacity (WHC) of fermented yogurt (over 77%). The Cata-lev treatment groups exhibited superior system stability (Zeta and WHC) than FOS. Overall, this study expanded knowledge about levan structure and its potential utilization as a yogurt stabilizer.

Crystal Structure of Levansucrase from the Gram-Negative Bacterium Brenneria Provides Insights into Its Product Size Specificity.

Microbial levansucrases (LSs, EC 2.4.1.10) have been widely studied for the synthesis of ß-(2,6)-fructans (levan) from sucrose. LSs synthesize levan-type fructo-oligosaccharides, high-molecular-mass levan polymer or combinations of both. Here, we report crystal structures of LS from the G--bacterium Brenneria sp. EniD 312 (Brs-LS) in its apo form, as well as of two mutants (A154S, H327A) targeting positions known to affect LS reaction specificity. In addition, we report a structure of Brs-LS complexed with sucrose, the first crystal structure of a G--LS with a bound substrate. The overall structure of Brs-LS is similar to that of G-- and G+-LSs, with the nucleophile (D68), transition stabilizer (D225), and a general acid/base (E309) in its active site. The H327A mutant lacks an essential interaction with glucosyl moieties of bound substrates in subsite +1, explaining the observed smaller products synthesized by this mutant. The A154S mutation affects the hydrogen-bond network around the transition stabilizing residue (D225) and the nucleophile (D68), and may affect the affinity of the enzyme for sucrose such that it becomes less effective in transfructosylation. Taken together, this study provides novel insights into the roles of structural elements and residues in the product specificity of LSs.

Exploiting pglB Oligosaccharyltransferase-Positive and -Negative Campylobacter jejuni and a Multiprotease Digestion Strategy to Identify Novel Sites Modified by N-Linked Protein Glycosylation.

Campylobacter jejuni is a bacterial pathogen encoding a unique N-linked glycosylation (pgl) system that mediates attachment of a heptasaccharide to N-sequon-containing membrane proteins by the PglB oligosaccharyltransferase (OST). Many targets of PglB are known, yet only a fraction of sequons are experimentally confirmed, and site occupancy remains elusive. We exploited pglB-positive (wild-type; WT) and -negative (ΔpglB) proteomes to identify potential glycosites. The nonglycosylated forms of known glycopeptides were typically increased in protein normalized abundance in ΔpglB relative to WT and restored by pglB reintroduction (ΔpglB::pglB). Sequon-containing peptide abundances were thus consistent with significant site occupancy in the presence of the OST. Peptides with novel sequons were either unaltered (likely not glycosylated) or showed abundance consistent with known glycopeptides. Topology analysis revealed that unaltered sequons often displayed cytoplasmic localization, despite originating from membrane proteins. Novel glycosites were confirmed using parallel multiprotease digestion, LC-MS/MS, and FAIMS-MS to define the glycoproteomes of WT and ΔpglB::pglBC. jejuni. We identified 142 glycosites, of which 32 were novel, and 83% of sites predicted by proteomics were validated. There are now 166 experimentally verified C. jejuni glycosites and evidence for occupancy or nonoccupancy of 31 additional sites. This study serves as a model for the use of OST-negative cells and proteomics for highlighting novel glycosites and determining occupancy in a range of organisms.

Active site variants in STT3A cause a dominant type I congenital disorder of glycosylation with neuromusculoskeletal findings.

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs.

Discovery of Glycosyltransferases Involved in the Biosynthesis of Ligupurpuroside B.

In this study, we report the characterization of three glycosyltransferases involved in the biosynthesis of ligupurpuroside B, a complex acylated phenolic glycoside in Ligustrum robustum. UGT85AF8 catalyzed the formation of salidroside from tyrosol. UGT79G7, an osmanthuside A 1,3-rhamnosyltransferase, and UGT79A19, an osmanthuside B 1,4-rhamnosyltransferase, sequentially converted osmanthuside A into ligupurpuroside B. Orthologs of UGT79G7 were also discovered from other plants producing verbascoside. These rhamnosyltransferases expand the toolbox for the biosynthesis of natural products with various sugar chains.

The structure of an archaeal oligosaccharyltransferase provides insight into the strict exclusion of proline from the N-glycosylation sequon.

Oligosaccharyltransferase (OST) catalyzes oligosaccharide transfer to the Asn residue in the N-glycosylation sequon, Asn-X-Ser/Thr, where Pro is strictly excluded at position X. Considering the unique structural properties of proline, this exclusion may not be surprising, but the structural basis for the rejection of Pro residues should be explained explicitly. Here we determined the crystal structure of an archaeal OST in a complex with a sequon-containing peptide and dolichol-phosphate to a 2.7 Å resolution. The sequon part in the peptide forms two inter-chain hydrogen bonds with a conserved amino acid motif, TIXE. We confirmed the essential role of the TIXE motif and the adjacent regions by extensive alanine-scanning of the external loop 5. A Ramachandran plot revealed that the ring structure of the Pro side chain is incompatible with the ϕ backbone dihedral angle around -150° in the rigid sequon-TIXE structure. The present structure clearly provides the structural basis for the exclusion of Pro residues from the N-glycosylation sequon.