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Enzyme clustering can improve catalytic efficiency by facilitating the processing of intermediates. Functional membrane microdomains (FMMs) in bacteria can provide a platform for enzyme clustering. However, the amount of FMMs at the cell basal level is still facing great challenges in multi-enzyme immobilization. Here, using the nutraceutical N-acetylglucosamine (GlcNAc) synthesis in Bacillus subtilis as a model, we engineered FMM components to improve the enzyme assembly in FMMs. First, by overexpression of the SPFH (stomatin-prohibitin-flotillin-HflC/K) domain and YisP protein, an enzyme involved in the synthesis of squalene-derived polyisoprenoid, the membrane order of cells was increased, as verified using di-4-ANEPPDHQ staining. Then, two heterologous enzymes, GlcNAc-6-phosphate N-acetyltransferase (GNA1) and haloacid dehalogenase-like phosphatases (YqaB), required for GlcNAc synthesis were assembled into FMMs, and the GlcNAc titer in flask was increased to 8.30 ± 0.57 g/L, which was almost three times that of the control strains. Notably, FMM component modification can maintain the OD600 in stationary phase and reduce cell lysis in the later stage of fermentation. These results reveal that the improved plasma membrane ordering achieved by the engineering FMM components could not only promote the enzyme assembly into FMMs, but also improve the cell fitness.

Bacillus subtilis PdaC (BsPdaC) is a membrane-bound, multidomain peptidoglycan N-deacetylase acting on N-acetylmuramic acid (MurNAc) residues and conferring lysozyme resistance to modified cell wall peptidoglycans. BsPdaC contains a C-terminal family 4 carbohydrate esterase (CE4) catalytic domain, but unlike other MurNAc deacetylases, BsPdaC also has GlcNAc deacetylase activity on chitooligosaccharides (COSs), characteristic of chitin deacetylases. To uncover the molecular basis of this dual activity, here we determined the X-ray structure of the BsPdaC CE4 domain at 1.54 Å resolution and analyzed its mode of action on COS substrates. We found that the minimal substrate is GlcNAc3 and that activity increases with the degree of glycan polymerization. COS deacetylation kinetics revealed that BsPdaC operates by a multiple-chain mechanism starting at the internal GlcNAc units and leading to deacetylation of all but the reducing-end GlcNAc residues. Interestingly, BsPdaC shares higher sequence similarity with the peptidoglycan GlcNAc deacetylase SpPgdaA than with other MurNAc deacetylases. Therefore, we used ligand-docking simulations to analyze the dual GlcNAc- and MurNAc-binding specificities of BsPdaC and compared them with those of SpPgdA and BsPdaA, representing peptidoglycan deacetylases highly specific for GlcNAc or MurNAc residues, respectively. BsPdaC retains the conserved Asp-His-His metal-binding triad characteristic of CE4 enzymes acting on GlcNAc residues, differing from MurNAc deacetylases that lack the metal-coordinating Asp residue. BsPdaC contains short loops similar to those in SpPgdA, resulting in an open binding cleft that can accommodate polymeric substrates. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases.

N-glycans are involved in various physiological functions and their structures diverge among different phyla and kingdoms. Insect cells mainly produce high mannose-type and paucimannose-type glycans but very few mammalian-like complex-type glycans. However, many insects possess genes for proteins homologous to the enzymes involved in complex-type N-glycan synthesis in mammalian cells, and their N-glycosylation pathway is incompletely understood compared with that of mammals. Here, we cloned a candidate gene for β-1,2-N-acetylglucosaminyltransferase II (GnTII), which is a Golgi-localized enzyme involved in a key step in the conversion to complex-type N-glycans, from silkworm Bombyx mori, and the gene was found to be expressed ubiquitously in the larval and pupal stages. In addition, recombinant B. mori GnTII was expressed as a soluble form using a silkworm-B. mori nucleopolyhedrovirus bacmid expression system. The recombinant enzyme exhibited similar pH and temperature dependency and the same substrate specificity as human GnTII, but deglycosylation with peptide:N-glycanase F did not affect its enzymatic activity. Compared with the structure of human GnTII, the amino acid residues involved in catalytic activity and substrate recognition are almost fully conserved in B. mori GnTII, which is consistent with its enzymatic properties. These results raised the possibility of mammalian-like complex-type N-glycan synthesis using the GnTII ortholog in silkworm.

A novel marine bacterium Arenibacter palladensis YHY2 isolated from Eastern Sea, South Korea revealed hydrolysing ability of crab chitin in shake flask and microbial fuel cell (MFC) systems. Under shake flask cultivation, strain YHY2 demonstrated 71.44 ± 1.90 U/ml chitinase activity with the microbial cell optical density (OD) 0.699 ± 0.021. High performance liquid chromatography (HPLC) results showed N-acetylglucosamine (GlcNAc) as the major by-product of chitin degradation. Furthermore, Ralstonia eutropha H16 which has carbon utilization limited to GlcNAc and fructose was co-cultivated with YHY2 to consume GlcNAc and accumulate polyhydroxyalkanoates (PHA). However, on co-cultivating strain YHY2 with H16 surprisingly showed higher production of chitinase (97.89 ± 2.72 U/ml) and GlcNAc (80.31 mg GlcNAc/g chitin) corresponding to the microbial cell OD (1.065 ± 0.005) at 120 h. In addition, marine strain YHY2 demonstrated biofilm formation, hence co-cultivation of strain YHY2 and H16 under MFC system was performed to check the electricity production. Maximum electricity current output density was 15.15 μA/cm2 at the initial stage and 10.72 μA/cm2 at 204 h. GlcNAc (196.34 mg GlcNAc/g chitin) was produced along with other metabolites like butyrate, acetate, lactate, and propionate in MFC system. The microbial cell biomass from MFC was analysed for PHA content showed 1.020 g/l of 3-polyhydroxybutyrate (PHB) and 0.198 g/l of 3-polyhydroxyvalerate (PHV) as the main components of PHA. Moreover, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) were performed to reveal changes in chitin before and after degradation. Thus, the exploitation of recalcitrant chitin biomass for simultaneous production of GlcNAc, electricity, and PHA in one-pot MFC system was the productive asset for sustainable development.

The marine sponge Amphimedon sp., collected from Hurghada (Egypt) was investigated for its sponge-derived actinomycetes diversity. Nineteen actinomycetes were cultivated and phylogenetically identified using 16S rDNA gene sequencing were carried out. The strains belong to genera Kocuria, Dietzia, Micrococcus, Microbacterium and Streptomyces. Many silent biosynthetic genes clusters were investigated using genome sequencing of actinomycete strains and has revealed in particular the genus Streptomyces that has indicated their exceptional capacity for the secondary metabolites production that not observed under classical cultivation conditions. In this study, the effect of N-acetylglucosamine on the metabolome of Streptomyces sp. RM66 was investigated using three actinomycetes media (ISP2, M1 and MA). In total, twelve extracts were produced using solid and liquid fermentation approaches. Liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data were analysed using metabolomics tools to compare natural product production across all crude extracts. Our study highlighted the elicitation effect of N-acetylglucosamine on the secondary metabolite profiles of Streptomyces sp. RM66. These results highlight the of N-acetylglucosamine application as an elicitor to induce the cryptic metabolites and for increasing the chemical diversity. All the twelve extracts were tested for their antibacterial activity was tested against Staphylococcus aureus NCTC 8325, antifungal activity against Candida albicans 5314 (ATCC 90028) and anti-trypanosomal activity against Trypanosoma brucei brucei. Extract St1 showed the most potent one with activities 2.3, 3.2 and 4.7 ug/ml as antibacterial, antifungal and anti-trypanosomal, respectively.

Chitin is the second most abundant carbohydrate biopolymer present in soils and is utilized by antibiotic-producing Streptomyces species. Its monomer, N-acetylglucosamine (GlcNAc), regulates the developmental program of the model organism Streptomyces coelicolor. GlcNAc blocks differentiation when growing on rich medium whilst it promotes development on poor culture media. However, it is unclear if the same GlcNAc regulatory profile observed in S. coelicolor applies also to other industrially important Streptomyces species. We report here the negative effect of GlcNAc on differentiation and tacrolimus (FK506) production by Streptomyces tsukubaensis NRRL 18488. Using microarrays technology, we found that GlcNAc represses the transcription of fkbN, encoding the main transcriptional activator of the tacrolimus biosynthetic cluster, and of ppt1, encoding a phosphopantheteinyltransferase involved in tacrolimus biosynthesis. On the contrary, GlcNAc stimulated transcription of genes related to amino acid and nucleotide biosynthesis, DNA replication, RNA translation, glycolysis and pyruvate metabolism. The results obtained support those previously reported for S. coelicolor, but some important differences were observed; for example genes involved in GlcNAc transport and metabolism and genes encoding transcriptional regulators such as crr, ptsI, nagE1, nagE2, nagB, chiA, chiJ, ngcE, dasR or atrA are not significantly induced in S. tsukubaensis by GlcNAc addition. Differences in the GlcNAc transport systems, in the physiology of S. tsukubaensis and S. coelicolor and/or the different composition of the culture media used are likely to be responsible for the discrepancies observed between these species.

N-glycans are highly crucial to the potential application prospect of glycoprotein. However, its structural diversity and instability severely limit its further research. Here we apply the tree-step computational approach to investigate preferred structures of disaccharides with β-1,4-linked N-acetylglucosamine and D-mannose of core pentasaccharide in N-glycans. Due to the structural character of disaccharides, hydrogen bonds (H-bonds) are classified according to the upper and lower parts of GlcNAcβ(1,4)GlcNAc and Manβ(1,4)GlcNAc. Almost all building conformers (95 for GlcNAcβ(1,4)GlcNAc and 50 for Manβ(1,4)GlcNAc) are generated depending on the combination of H-bonds in the upper and lower parts, except for one conformer. After establishing initial configurations, theoretical simulations for optimization, preferred structures and frequency calculations are employed. Both energy and IR vibration signature are in good accord with the experimental and theoretical literature. Additionally, five new low-energy structures are found, which have not been reported by using the traditional conformational search.

In previous work, a recombinant Bacillus subtilis strain was successfully constructed for microbial production of N-acetylglucosamine (GlcNAc). In this study, GlcNAc titer was further improved by combinatorial promoter engineering of key genes glck encoding glucokinase and pgi encoding phosphoglucoisomerase. First, three promoters including constitutive promoter P43, xylose inducible promoter PxylA, and isopropyl-β-d-thiogalactoside inducible Pgrac were used to replace the native promoters of glcK and pgi, yielding 12 recombinant strains. It was found that when glcK and pgi were both under the control of promoter PxylA, the highest GlcNAc titer in 3-L fed-batch bioreactor reached 35.12g/L, which was 52.6% higher than that of the initial host. Next, the transcriptional levels of the related genes in glycolysis, GlcNAc synthesis pathway, peptidoglycan synthesis pathway, and pentose phosphate pathway were investigated by quantitative real-time PCR analysis. Fine-tuning upper GlcNAc synthesis pathway by combinatorial promoter substitution significantly enhanced GlcNAc production in engineered B. subtilis.

Complex formation of D-glucosamine (Gl) and N-acetyl-D-glucosamine (AGl) with capsaicin (Cp) were studied by 1H NMR titrations in H2O-d2 and DMSO-d6; capsaicin is the major bioactive component of chili peppers. Every titration curve has been interpreted by formulating a suitable model for the reaction equilibrium, to elucidate intermolecular interactions. In DMSO, glucosamine cations associate with each other to yield linear aggregates, and undergo pseudo-1:1-complexation with capsaicin, the formation constant being ca. 30 M−1. N-Acetylglucosamine, without self-association, forms a 2:1-complex AGl2Cp with the stability of ca. 70 M−2. These complexations are achieved by intermolecular hydrogen bonds. In D2O, glucosamine undergoes reversible protonation equilibrium between Gl0 and GlH+ with the logarithmic protonation constants log KD = 8.63 for α-glucosamine and 8.20 for β-isomer. Both anomeric isomers of deprotonated glucosamine form Gl0Cp-type complexes of capsaicin, in a competitive manner, with a formation constant of 1040 M−1 for the α-glucosamine complex and 830 M−1 for the β-complex; the anomeric carbons result in the difference in thermodynamic stability. The reactant molecules are closed up by the solvent-exclusion effect and/or the van der Waals interaction; the resulting pair is stabilized by intermolecular hydrogen bonding within a local water-free space between the component molecules. By contrast, neither protonated glucosamine (GlH+) nor N-acetylglucosamine yields a capsaicin complex with the definite stoichiometry. The monosaccharides recognize capsaicin under only a controlled condition; the same phenomena are predicted for biological systems and nanocarriers based on polysaccharides such as chitosan.

In many Lactobacillales species (i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen,Streptococcus pyogenes, synthesizes a key antigenic surface polymer—the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N-acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphorylundecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltranferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function inStreptococcus agalactiaeandEnterococcus faecalis. In conclusion, the elucidation of GAC biosynthesis inS. pyogenesreported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall.

Intracellular GlcNAcylation of Ser and Thr residues is a well-known and widely investigated post-translational modification. This post-translational modification has been shown to play a significant role in cell signaling and in many regulatory processes within cells. O-GlcNAc transferase is the enzyme responsible for glycosylating cytosolic and nuclear proteins with a single GlcNAc residue on Ser and Thr side-chains. Here we report that the same enzyme may also be responsible for S-GlcNAcylation, i.e. for linking the GlcNAc unit to the peptide by modifying a cysteine side-chain. We also report that O-GlcNAcase, the enzyme responsible for removal of O-GlcNAcylation does not appear to remove the S-linked sugar. Such Cys modifications have been detected and identified in mouse and rat samples. This work has established the occurrence of 14 modification sites assigned to 11 proteins unambiguously. We have also identified S-GlcNAcylation from human Host Cell Factor 1 isolated from HEK-cells. Although these site assignments are primarily based on electron-transfer dissociation mass spectra, we also report that S-linked GlcNAc is more stable under collisional activation than O-linked GlcNAc derivatives.

(d)-Glucosamine and other nutritional supplements have emerged as safe alternative therapies for osteoarthritis, a chronic and degenerative articular joint disease. N-acetyl-(d)-glucosamine, a compound that can be modified at the N position, is considered to improve the oral bioavailability of (d)-glucosamine and has been proven to possess greater in vitro chondroprotective activity compared with the parent agent. In this study, to further utilize these properties, we focus on the modification of the N position with a benzenesulfonyl and different isoxazole formyl groups. Among these compounds, the 3-(2-chlorobenzene)-5-methyl-isoxazole formyl chloride and p-methoxybenzenesulfonyl chloride modifying structures proved to be the most active of the series and efficiently processed the chondrocytes in vitro. These novel N-position substitution compounds may represent promising leads for osteoarthritis drug development.

The exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm-dependent bacterial infections. The cationic charge resulting from partial de-N-acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity on de-N-acetylated PNAG oligosaccharides, but the molecular basis for this increased activity is not known. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site-directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the -1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG-dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.

As a traditional amino acid producing bacterium, Corynebacterium glutamicum is a platform strain for production of various fine chemicals. Based on the CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 system, gene editing tools that enable base conversion in the genome of C. glutamicum have been developed. However, some problems such as genomic instability caused by DNA double-strand break (DSB) and off-target effects need to be solved. In this study, a DSB-free single nucleotide genome editing system was developed by construction of a bi-directional base conversion tool TadA-dCas9-AID. This system includes cytosine base editors (CBEs): activation-induced cytidine deaminase (AID) and adenine deaminase (ABEs): tRNA adenosine deaminase (TadA), which can specifically target the gene through a 20-nt single guide RNA (sgRNA) and achieve the base conversion of C-T, C-G and A-G in the 28-bp editing window upstream of protospacer adjacent motif. Finally, as a proof-of-concept demonstration, the system was used to construct a mutant library of zwf gene in C. glutamicum S9114 genome to improve the production of a typical nutraceutical N-acetylglucosamine (GlcNAc). The GlcNAc titer of the mutant strain K293R was increased by 31.9% to 9.1 ​g/L in shake flask. Here, the developed bases conversion tool TadA-dCas9-AID does not need DNA double-strand break and homologous template, and is effective for genome editing and metabolic engineering in C. glutamicum.

Chitin represents the second most abundant biomass after lignocelluloses in the biosphere. It can be depolymerized into either N-acetylglucosamine (GlcNAc) or glucosamine (GlcN) and acetate by different degradation strategies. However, these chitin-derived carbon sources have been scarcely compared for lipid production. Here, GlcNAc was found superior to GlcN or acetate for lipid accumulation by Cutaneotrichosporon oleaginosum. The lipid accumulation potential of these carbon sources was calculated based on a small scale metabolic model of C. oleaginosum. Co-fermentation of GlcN and acetate under phosphate limitation rendered improved lipid production. GlcN and acetate were assimilated simultaneously. The highest lipid titer and yield of 10.1 g/L and 0.25 g/g, respectively, was reached when GlcNAc was used under phosphate limitation. The fatty acids composition of the lipid samples showed similarities to vegetable oils, demonstrating the suitability in biodiesel industry. This study provides profitable guidance for the design of chitin-to-lipids routes.

N-acetylglucosamine analogues exist in nature as an important saccharide in human body and can regulate a variety of life activities captured through biological nanochannels. Herein we describe a biomimetic strategy via supramolecular host-guest chemistry for constructing artificial nanochannels to detect N-acetylglucosamine analogues. The functional nanochannels exhibited a high selectivity and good reversibility towards N-acetylglucosamine. Further studies indicate that the detecting behaviors was probably due to the multiple hydrogen bonding interactions and an appropriately fitting the functional and geometric requirements between the tryptophan-decorated pillararenes and N-acetylglucosamine, leading to the change of the surface charge and wettability within the nanochannels. These studies can give interesting insight for the research of biological processes caused by N-acetylglucosamine-sensitive ion channels, and also help to develop the platform for detecting N-acetylglucosamine in vitro.

Glycosylation is a critical quality attribute of many therapeutic proteins, particularly monoclonal antibodies (MAbs). Nucleotide-sugar precursors supplemented to growth medium to affect the substrate supply chain of glycosylation has yielded promising but varied results for affecting glycosylation. Glucosamine (GlcN), a precursor for N-acetylglucosamine (GlcNAc), is a major component of mammalian glycans. The supplementation of GlcN to CHO cells stably-expressing a chimeric heavy-chain monoclonal antibody, EG2-hFc, reduces the complexity of glycans to favour G0 glycoforms, while also negatively impacting cell growth. Although several researchers have examined the supplementation of glucosamine, no clear explanation of its impact on cell growth has been forthcoming. In this work, the glucosamine metabolism is examined. We identified the acetylation of GlcN to produce GlcNAc to be the most likely cause for the negative impact on growth due to the depletion of intracellular acetyl-CoA pools in the cytosol. By supplementing GlcNAc in lieu of GlcN to CHO cells producing EG2-hFc, we achieve the same shift in glycan complexity with marginal impacts on the cell growth and protein production.

The most abundant N-glycan in plants is the paucimannosidic N-glycan with core β1,2-xylose and α1,3-fucose residues (Man3XylFuc(GlcNAc)2). Here, we report a mechanism in Arabidopsis thaliana that efficiently produces the largest N-glycan in plants. Genetic and biochemical evidence indicates that the addition of the 6-arm β1,2-GlcNAc residue by N-acetylglucosaminyltransferase II (GnTII) is less effective than additions of the core β1,2-xylose and α1,3-fucose residues by XylT, FucTA, and FucTB in Arabidopsis. Furthermore, analysis of gnt2 mutant and 35S:GnTII transgenic plants shows that the addition of the 6-arm non-reducing GlcNAc residue to the common N-glycan acceptor GlcNAcMan3(GlcNAc)2 inhibits additions of the core β1,2-xylose and α1,3-fucose residues. Our findings indicate that plants limit the rate of the addition of the 6-arm GlcNAc residue to the common N-glycan acceptor as a mechanism to facilitate formation of the prevalent N-glycans with Man3XylFuc(GlcNAc)2 and (GlcNAc)2Man3XylFuc(GlcNAc)2 structures.

Background O-linked β- N -acetylglucosamine (O-GlcNAc) modification of epidermal growth factor (EGF) domains catalyzed by EGF domain O-GlcNAc transferase (EOGT) is the first example of GlcNAc modification in the lumen of the endoplasmic reticulum (ER). Scope of review This review summarizes current knowledge on the EOGT-catalyzed O-GlcNAc modification of EGF domains obtained through biochemical characterization, genetic analysis in Drosophila , and identification of human EOGT mutation. Additionally, this review discusses GTDC2—another ER protein homologous to EOGT that catalyzes the GlcNAc modification of O-mannosylated α-dystroglycan—and other components of the biosynthetic pathway involved in GlcNAc modification in the ER lumen. Major conclusions GlcNAc modification in the ER lumen has been identified as a novel type of protein modification that regulates specific protein function. Moreover, abnormal GlcNAc modification in the ER lumen is responsible for Adams–Oliver syndrome and Walker–Warburg syndrome. General significance Elucidation of the biological function of GlcNAc modification in the ER lumen will provide new insights into the unique roles of O-glycans, whose importance has been demonstrated in multifunctional glycoproteins such as Notch receptors and α-dystroglyan.

In our previous work, a recombinant Bacillus subtilis strain for the microbial production of N-acetylglucosamine (GlcNAc) was constructed through modular pathway engineering. In this study, to enhance GlcNAc production, glucose feeding approaches and dissolved oxygen (DO) control methods in fed-batch culture were systematically investigated. We first studied the effects of different glucose feeding strategies, including exponential fed-batch culture, pulse fed-batch culture, constant rate fed-batch culture, and glucose control (5 g/L, 10 g/L, 15 g/L) fed-batch culture, on cell growth and GlcNAc synthesis. We found that GlcNAc production in glucose control (5 g/L) fed-batch culture reached 26.58 g/L, which was 3.10 times that in batch culture. Next, the effect of DO level (20%, 30%, 40%, and 50%) on GlcNAc production was investigated, and a step-wise DO control strategy (0-7 h, 30%; 7-15 h, 50%; 15-50 h, 40%; 50-72 h, 30%) was introduced. With the optimal glucose and DO control strategy, GlcNAc production reached 35.77 g/L, which was 4.17 times the production in batch culture without DO control.

Streptococcus iniae infection has emerged as a serious fish health and economic problem in the global aquaculture operations. Current antibiotic options are few and possess severe practical limitations and potential adverse environmental impacts. The major factor contributing to the large burden of S. iniae disease in aquaculture is the lack of fundamental knowledge of innate immunity against the pathogen. In the present study, we use a tilapia model to explore which metabolites are crucial for the defense against the infection caused by S. iniae. We establish GC/MS based metabolic profile of tilapia liver and then compare the metabolic difference between survivals and the dying fish post the bacterial infection. We identify elevating N-acetylglucosamine in survival group as the most crucial metabolite differentiating the survivals from the dying in these fish infected by S. iniae. Exogenous N-acetylglucosamine significantly elevates survival ability of tilapia against the infection caused by S. iniae. Our findings highlight the importance of metabolic strategy against bacterial infections.

An endochitinase and β-N-acetylglucosaminidase (NAGase) were purified and characterised from fresh rubber latex serum. These enzymes were used in a total enzyme-based system to produce pure N-acetylglucosamine (NAG) from chitin. The N-terminal amino acid sequences of both purified endochitinase (KEESRRRRHR) and NAGase (AAVDSDTLEI) lacked homology with other known chitinases, including hevamine from rubber latex lutoids. The apparent kinetic parameters, Km and Vmax, for the endochitinase using 4-MU-β-(NAG)3 as a substrate were 99.73 μM and 29.49 pkat mg(-1), respectively. For NAGase, using 4-MU-β-NAG as a substrate, the corresponding Km and Vmax values were 20.4 μM and 25.82 pkat mg(-1). When an enzyme incubation mixture containing a 1:1 (pkat/pkat) activity mixed ratio of endochitinase: NAGase was employed, the maximum yield of N-acetylglucosamine (NAG) obtained was 98% from β-chitin and 20% from α-chitin. These yields were obtained after 4 days of hydrolysis of equal amounts of β-chitin and α-chitin in the mixture. Thus, β-chitin was the preferred substrate compared to α-chitin by a ratio of nearly five to one. Mass spectroscopic analysis, using electrospray ionisation mass spectrometry (ESI-MS), of the product obtained from β-chitin after digestion (for 24h) depicted one distinct major molecular ion peak m/z 260.1, a small minor ion peak m/z 481.2, a potassium adduct of NAG and a potassium adduct of two NAG molecules. Furthermore, experiments to establish the commercial production of NAG using crude enzymes of Hevea latex serum are currently in progress.

33 p.-6 fig.-1 tab.-1 schem.-1 graph. abst, An unprecedented approach towards oligosaccharides containing N-acetylglucosamine-N-acetylmuramic (NAG-NAM) units was developed. These novel bacterial cell wall surrogates were obtained from chitosan via a top down approach involving both chemical and enzymatic reactions. The chemical modification of chitosan using a molecular clamp based strategy, allowed obtaining N-acetylglucosamine-N-acetylmuramic (NAG-NAM) containing oligomers. Intercalation of NAM residues was confirmed through the analysis of oligosaccharide fragments from enzymatic digestion and it was found that this route affords NAG-NAM containing oligosaccharides in 33% yield. These oligosaccharides mimic the carbohydrate basic skeleton of most bacterial cell surfaces. The oligosaccharides prepared are biologically relevant and will serve as a platform for further molecular recognition studies with different receptors and enzymes of both bacterial cell wall and innate immune system. This strategy combining both chemical modification and enzymatic digestion provides a novel and simple route for an easy access to bacterial cell wall fragments – biologically important targets., This work was funded by Fundação para a Ciência e Tecnologia (FC&T) for: fellowships SFRH/BD/89518/2012, SFRH/BD/52207/2013 (to FQ and GC); projects (PTDC/QEQ-QOR/2132/2012 and UID/DTP/04138/2013). This work was supported by the Associate Laboratory for Green Chemistry- LAQV which is financed by national funds from FCT/MCTES (UID/QUI/50006/2019) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER - 007265). i3N|Cenitmat is financed by national funds from FC&T/MEC (UID/CTM/50025/2019) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007688) The National NMR Facility is supported by FC&T (ROTEIRO/0031/2013 – PINFRA/22161/2016, co-financed by FEDER through COMPETE 2020, POCI, and PORL and FC&T through PIDDAC). iNOVA4 Health Research Unit (LISBOA-01-0145-FEDER-007344), is co-funded by FC&T/Ministério da Ciência e do Ensino Superior, and by FEDER under the PT2020 Partnership Agreement. CTQ2012‐32025 and CTQ2015-64597-C2 of Spanish Ministry of Economy and Competitiveness to FJC are acknowledge. The LC–MS/MS equipment is part of The National MS Facility, supported by FC&T (REDE/1518/REM/2005). This work was supported by the Applied Molecular Biosciences Unit-UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2019).

Dendritic cell inhibitory receptor 2 (DCIR2) is a C-type lectin expressed on classical dendritic cells. We recently identified the unique ligand specificity of mouse DCIR2 (mDCIR2) toward biantennary complex-type glycans containing bisecting N-acetylglucosamine (GlcNAc). Here, we report the crystal structures of the mDCIR2 carbohydrate recognition domain in unliganded form as well as in complex with an agalactosylated complex-type N-glycan unit carrying a bisecting GlcNAc residue. Bisecting GlcNAc and the α1-3 branch of the biantennary oligosaccharide asymmetrically interact with canonical and non-canonical mDCIR2 residues. Ligand-protein interactions occur directly through mDCIR2-characteristic amino acid residues as well as via a calcium ion and water molecule. Our structural and biochemical data elucidate for the first time the unique binding mode of mDCIR2 for bisecting GlcNAc-containing glycans, a mode that contrasts sharply with that of other immune C-type lectin receptors such as DC-SIGN.

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.

Chitin, which is a polymer of β-(1–4) linked N-acetyl- d -glucosamine (GlcNAc) residues, is one of the most abundant renewable resources in nature, after cellulose. In this study, we found some native Mucor strains, which can use GlcNAc and chitin substrates as carbon sources for growth and ethanol production. One of these strains, M. circinelloides NBRC 6746 produced 18.6 ± 0.6 g/l of ethanol from 50 g/l of GlcNAc after 72 h and the maximum ethanol production rate was 0.75 ± 0.1 g/l/h. Furthermore, M. circinelloides NBRC 4572 produced 6.00 ± 0.22 and 0.46 ± 0.04 g/l of ethanol from 50 g/l of colloidal chitin and chitin powder after 16 and 12 days, respectively. We also found an extracellular chitinolytic enzyme producing strain M. ambiguus NBRC 8092, and successfully improved ethanol productivity of NBRC 4572 from colloidal chitin using crude chitinolytic enzyme derived from NBRC 8092. The ethanol titer reached 9.44 ± 0.10 g/l after 16 days. These results were the first bioethanol production from GlcNAc and chitin substrates by native organisms, and also suggest that these Mucor strains have great potential for the simultaneous saccharification and fermentation (SSF) of chitin biomass.

Synthesis of N-acetylneuraminic acid (Neu5Ac) from N-acetylglucosamine (GlcNAc) and pyruvate was carried out by constructing and expressing a polycistronic plasmid encoding an N-acetylglucosamine 2-epimerase (AGE) gene and an N-acetylneuraminic acid lyase (Nal) gene simultaneously. Nal from Escherichia coli K12 and AGEs from Synechocystis sp. PCC 6803 (snAGE) and Anabaena sp. CH1 (anAGE) were used. And four polycistronic plasmids were constructed in which the positions of AGE gene differed with respect to Nal gene. Among these plasmids, pET-28a-Nal-anAGE with anAGE gene located next to Nal gene caused the production of the highest amount of Neu5Ac, generating 61.3g/L in 60h by whole-cell catalysis without the addition of ATP as AGE activator. And pET-28a-Nal-anAGE lowered anAGE's expression level, allowing it to fold properly. Thus, an inclusion-body-free E. coli strain capable of producing Neu5Ac by whole-cell catalysis with high yield and low cost was constructed in the present study.

A previously described aryl sulfonamide series, originally found through HTS, targets GlmU, a bifunctional essential enzyme involved in bacterial cell wall synthesis. Using structure-guided design, the potency of enzyme inhibition was increased in multiple isozymes from different bacterial species. Unsuitable physical properties (low LogD and high molecular weight) of those compounds prevented them from entering the cytoplasm of bacteria and inhibiting cell growth. Further modifications described herein led to compounds that possessed antibacterial activity, which was shown to occur through inhibition of GlmU. The left-hand side amide and the right-hand side sulfonamides were modified such that enzyme inhibitory activity was maintained (IC(50)0.1 μM against GlmU isozymes from Gram-negative organisms), and the lipophilicity was increased giving compounds with LogD -1 to 3. Antibacterial activity in an efflux-pump deficient mutant of Haemophilus influenzae resulted for compounds such as 13.

We describe the improvement of transglycosylation (TG) by chitinase D from Serratia proteamaculans (SpChiD). The SpChiD produced a smaller quantity of TG products for up to 90 min with 2 mm chitotetraose as the substrate and subsequently produced only hydrolytic products. Of the five residues targeted at the catalytic center, E159D resulted in substantial loss of both hydrolytic and TG activities. Y160A resulted in a product profile similar to SpChiD and a rapid turnover of substrate with slightly increased TG activity. The rest of the three mutants, M226A, Y228A, and R284A, displayed improved TG and decreased hydrolytic ability. Four of the five amino acid substitutions, F64W, F125A, G119S, and S116G, at the catalytic groove increased TG activity, whereas W120A completely lost the TG activity with a concomitant increase in hydrolysis. Mutation of Trp-247 at the solvent-accessible region significantly reduced the hydrolytic activity with increased TG activity. The mutants M226A, Y228A, F125A, S116G, F64W, G119S, R284A, and W247A accumulated approximately double the concentration of TG products like chitopentaose and chitohexaose, compared with SpChiD. The double mutant E159D/F64W regained the activity with accumulation of 6.0% chitopentaose at 6 h, similar to SpChiD at 30 min. Loss of chitobiase activity was unique to Y228A. Substitution of amino acids at the catalytic center and/or groove substantially improved the TG activity of SpChiD, both in terms of the quantity of TG products produced and the extended duration of TG activity.

International audience; Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg2+ ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.

Human milk contains high amounts of complex oligosaccharides, which can be utilized especially by Bifidobacterium species in the infant gut as a carbon and energy source. N-acetyl-d-glucosamine is a building block of these oligosaccharides, and molecular details on the release and utilization of this monosaccharide are not fully understood. In this work we have studied some of the enzymatic properties of three N-acetyl-β-D-hexosaminidases encoded by the genome of the intestinal isolate Bifidobacterium longum subsp. infantis ATCC 15697 and the gene expression of the corresponding genes during bacterial growth on human milk oligosaccharides. These enzymes belong to the glycosyl hydrolase family 20, with several homologs in bifidobacteria. Their optimum pH was 5.0 and optimum temperature was 37 °C. The three enzymes were active on the GlcNAcβ1-3 linkage found in lacto-N-tetraose, the most abundant human milk oligosaccharide. Blon_0459 and Blon_0732, but not Blon_2355, cleaved branched GlcNAcβ1-6 linkages found in lacto-N-hexaose, another oligosaccharide abundant in breast milk. Bifidobacterium infantis N-acetyl-β-D-hexosaminidases were induced during early growth in vitro on human milk oligosaccharides, and also during growth on lacto-N-tetraose or lacto-N-neotetraose. The up-regulation of enzymes that convert this monosaccharide into UDP-N-acetylglucosamine by human milk oligosaccharides suggested that this activated sugar is used in peptidoglycan biosynthesis. These results emphasize the complexity of human milk oligosaccharide consumption by this infant intestinal isolate, and provide new clues into this process.

The terminal modification of glycans by β4 addition of N-acetylgalactosamine to N-acetylglucosamine with formation of the N,N-diacetyllactosediamine (LacdiNAc) moiety has been well documented for a number of N-linked glycoproteins and peptides, like neurohormones. Much less is known about O-glycoproteins in this regard because only human zona pellucida glycoprotein 3 (ZP3) and bovine proopiomelanocortin were reported to be LacdiNAc-modified. In searching for mammalian proteins modified with O-linked LacdiNAc we identified six positive species among nine endogenous and recombinant O-glycoproteins, which were extracellular matrix, or matrix-related proteins. These are ZP3 and the five novel LacdiNAc-positive species ECM1, AMACO, nidogen-1, α-dystroglycan, and neurofascin. The mass spectrometric analyses revealed a core 2-based tetrasaccharide as the common structural basis of O-linked LacdiNAc that could be further modified, similar to the type 2 LacNAc termini, with fucose, sialic acid, or sulfate. Here, we provide structural evidence for a novel type of mucin-type O-glycans that is strictly specific for LacdiNAc termini: sugar phosphorylation with formation of GalNAcβ1-4(phospho-)GlcNAc. The structural details of the phosphatase-labile compound were elucidated by MS(2) analysis of tetralysine complexes and by MS(n) measurements of the permethylated glycan alditols. Phospho-LacdiNAc was detected in human HEK-293 as well as in mouse myoblast cells and in bovine brain tissue.

It is necessary to develop highly functionalized liver cell culture systems for liver tissue engineering such as bioartificial livers and liver cell chips. To maintain a high level of hepatocyte function, well-organized patterning culture systems of hepatocytes and nonparenchymal cells would be advantageous. To design the patterning culture system using these cells, cell-recognizable polymers should be useful to regulate not only the hepatocytes, but also the nonparenchymal cells. Here, we report that N-acetylglucosamine (GlcNAc)-bearing polymers are useful as nonparenchymal cell-recognizable polymers. It has previously been reported that mesenchymal cells adhered to GlcNAc-bearing polymer-coated dishes through surface vimentin. It was also observed that nonparenchymal cells expressing vimentin or desmin specifically adhered to GlcNAc-bearing polymer-coated dishes. Especially, in hepatic stellate cells (HSCs) cultured on GlcNAc-bearing polymer-coated dishes, the expression of α-smooth muscle actin as an activated HSCs marker was suppressed in long-term. Therefore, HSCs were shown to maintain a quiescent state on PVGlcNAc-coated dishes during a long-term culture. These results demonstrated that GlcNAc-bearing polymers could be beneficial to culture nonparenchymal cells such as HSCs. Our findings suggest that galactose- and GlcNAc-bearing polymers can regulate the culture of all liver cells and may be useful tools for the establishment of liver tissue engineering.

The extracellular parasite Trichomonas vaginalis contains a surface glycoconjugate that appears to mediate parasite-host cell interaction via binding to human galectin-1. This glycoconjugate also elicits cytokine production from human vaginal epithelial cells, implicating its role in modulation of host immune responses. We have analyzed the structure of this glycoconjugate, previously described to contain the sugars rhamnose (Rha), N-acetylglucosamine (GlcNAc), galactose (Gal), xylose (Xyl), N-acetylgalactosamine (GalNAc), and glucose (Glc), using gas chromatograph mass spectrometry (GC-MS), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF), electrospray MS/MS, and nuclear magnetic resonance (NMR), combined with chemical and enzymatic digestions. Our data reveal a complex structure, named T. vaginalis lipoglycan (TvLG), that differs markedly from Leishmania lipophosphoglycan and Entamoeba lipopeptidophosphoglycan and is devoid of phosphosaccharide repeats. TvLG is composed of an α1–3 linked polyrhamnose core, where Rha residues are substituted at the 2-position with either β-Xyl or chains of, on average, five N-acetyllactosamine (-3Galβ1–4GlcNAcβ1-) (LacNAc) units and occasionally lacto-N-biose (-3Galβ1-3GlcNAcβ1-) (LNB). These chains are themselves periodically substituted at the Gal residues with Xyl-Rha. These structural analyses led us to test the role of the poly-LacNAc/LNB chains in parasite binding to host cells. We found that reduction of poly-LacNAc/LNB chains decreased the ability of TvLG to compete parasite binding to host cells. In summary, our data provide a new model for the structure of TvLG, composed of a polyrhamnose backbone with branches of Xyl and poly-LacNAc/LNB. Furthermore, the poly-LacNAc side chains are shown to be involved in parasite-host cell interaction.

Peptidoglycan (PG) N-acetyl muramic acid (MurNAc) O-acetylation is widely spread in gram-positive bacteria and is generally associated with resistance against lysozyme and endogenous autolysins. We report here the presence of O-acetylation on N-acetylglucosamine (GlcNAc) in Lactobacillus plantarum PG. This modification of glycan strands was never described in bacteria. Fine structural characterization of acetylated muropeptides released from L. plantarum PG demonstrated that both MurNAc and GlcNAc are O-acetylated in this species. These two PG post-modifications rely on two dedicated O-acetyltransferase encoding genes, named oatA and oatB, respectively. By analyzing the resistance to cell wall hydrolysis of mutant strains, we showed that GlcNAc O-acetylation inhibits N-acetylglucosaminidase Acm2, the major L. plantarum autolysin. In this bacterial species, inactivation of oatA, encoding MurNAc O-acetyltransferase, resulted in marked sensitivity to lysozyme. Moreover, MurNAc over-O-acetylation was shown to activate autolysis through the putative N-acetylmuramoyl-L-alanine amidase LytH enzyme. Our data indicate that in L. plantarum, two different O-acetyltransferases play original and antagonistic roles in the modulation of the activity of endogenous autolysins.

The production of N-acetylglucosamine (GlcNAc, chitin monosaccharide) from crab or shrimp shells generally requires numerous steps and the use of deleterious substances. One goal for researchers is the direct production of GlcNAc and NN′-diacetylchitobiose [(GlcNAc)2, chitin structural dimeric unit] from both chitin and crab shells. The present study reports the development of an intensive ball “converge” mill for the rapid mechanochemical conversion of chitin or crab shells into amorphous, chitinase-sensitive microparticles. Optimal crab shell grinding parameters were determined, and close to 100% direct degradation of chitin from crab shell to GlcNAc was achieved. This is the first report of using a mechanochemical process with enzymatic degradation to decrease the environmental burdens associated with GlcNAc production from chitin.

An efficient synthesis of sialyllactosamine (SiaLacNAc) clusters using carbosilanes as core scaffolds has been accomplished by means of chemical and enzymatic approaches. N -Acetyl- d -glucosamine (GlcNAc) clusters having O -glycosidic linkage or S -glycosidic linkage were chemically synthesized from known intermediates in high yields. The GlcNAc clusters were first used as substrates for β1,4 galactosyl transferase using UDP-galactose (UDP-Gal) as a sugar source to provide corresponding N -acetyllactosamine clusters. Further sugar elongation of the LacNAc clusters was demonstrated using α2,3 sialyl transferase and CMP-neuraminic acid (CMP-NANA) to yield the corresponding SiaLacNAc clusters.

A chitinase (CHT), a chitosanase (CHS) and a protease (PRO) were purified from the culture supernatant of Serratia sp. TKU020 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHT, CHS and PRO determined by SDS-PAGE were approximately 65 kDa, 55 kDa and 55 kDa, respectively. CHT and CHS were inhibited by Mn 2+ , EDTA and PRO was inhibited by Mg 2+ , EDTA. The antioxidant activity of TKU020 culture supernatant was 78% (DPPH scavenging ability). N -Acetylglucosamine (GlcNAc) and N -acetyl chitobiose (GlcNAc) 2 were also produced from the culture supernatant by using TKU020 strain fermentation. The maximum production of GlcNAc and (GlcNAc) 2 was 1.3 mg/mL and 2.7 mg/mL, respectively, after 4 days of fermentation. With this method, we have shown that squid pen wastes can be utilized and it is effective in the production of enzymes, antioxidants, and N -acetyl chitooligosaccharides, facilitating its potential use in industrial applications and functional foods.

Mutant glycosynthases or transglycosidases obtained from a Thermus thermophilus β- d -glycosidase (TtbGly) efficiently catalyzed the synthesis of β-(1→3)-disaccharides. Unfortunately, this regioselectivity was changed to the β-(1→4) one when N-acetylglucosamine derivatives were used as acceptors, thus precluding the possibility of synthesizing d -Galp-β-(1→3)- d -GlcpNAc (lacto-N-biose) or d -Glcp-β-(1→3)- d -GlcpNAc, which are useful synthons for the synthesis of antigen determinants. In contrast, we show in this work that, in the presence of phenyl 2-amino-1-thio-β- d -glucopyranoside, the ‘normal’ β-(1→3) regioselectivity of E338G TtbGly glycosynthase takes place. Thus, transglycosylations using α-galactosyl or α-glucosyl fluorides gave the corresponding phenyl β- d -glycopyranosyl-(1→3)-2-amino-2-deoxy-1-thio-β- d -glucopyranosides in high yields (88–97%). Subsequent selective N-acylation followed by NBS/water deprotection of the thiophenyl group afforded lacto-N-biose in high overall yields.

A wide variety of medically important biofilm forming bacteria produce similar polysaccharide intracellular adhesins (PIAs). The PIA structures consist of partially de-N-acetylated β-(1→6)-N-acetylglucosamine polymers. These exopolysaccharides are key components of the bacterial biofilm matrix. Here, we describe the efficient synthesis of PIA oligosaccharides using an acid reversion reaction of N-acetylglucosamine in HF·pyridine. The PIA oligosaccharides produced by this reaction can be purified to homogeneity by size exclusion chromatography. Chemistry was developed to conjugate the PIA oligosaccharides to bovine serum albumin using a new heterobifunctional linker containing a thiol and an N-methylhydroxylamine functional group. These glycoconjugates may serve as useful precursors for the development of defined conjugate vaccines against PIA producing bacterial strains.

Ficolins are collagenous lectins that bind N-acetylated glycans and participate in innate immune responses, including phagocytosis and complement activation. Related collagenous lectins such as mannan binding lectin (MBL) and surfactant proteins A and D possess antiviral activity, but this activity has not been demonstrated for ficolins. In these studies, we used purified porcine plasma ficolin alpha and recombinant ficolin alpha to assess their ability to bind and neutralize porcine reproductive and respiratory virus (PRRSV) in various assays. Recombinant ficolin alpha was designed with a C-terminal 6-histidine tag using a pcDNA3.1 expression vector system in CHO K1 cells. Plasma-purified and recombinant ficolin alpha reduced cytopathic effect of PRRSV-infected Marc-145 cells in neutralization assays and inhibited replication of infectious viral particles in a GlcNAc-dependent manner. In vitro replication determined by plaque assay was inhibited in the presence of plasma-purified ficolin alpha and recombinant ficolin. Immunoreactive plasma ficolin alpha and recombinant ficolin alpha also bound PRRSV-coated wells in a GlcNAc-dependent manner. These studies indicate that porcine ficolin can bind and neutralize a common arterivirus that is a major pathogen of swine.

Two tigogenyl glycosides containing N-acetylglucosamine were synthesized. Their structures were confirmed by 1H and 13C NMR spectra. The shielding effect caused by benzoyl groups was elucidated by 1H NMR, COSY, HSQC, HMBC spectroscopy.

Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a beta-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a beta-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the beta(1,2)-linked N-acetylglucosamine from the Man alpha(1,3) branch compared with the Man alpha(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal beta-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing beta-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.

N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthetic process of UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a UDP sugar that serves as a biosynthetic precursor of glycoproteins, mucopolysaccharides, and the cell wall of bacteria. Thus, a specific inhibitor of AGM1 from pathogenetic fungi could be a new candidate for an antifungal reagent that inhibits cell wall synthesis. AGM1 catalyzes the conversion of N-acetylglucosamine 6-phosphate (GlcNAc-6-P) into N-acetylglucosamine 1-phosphate (GlcNAc-1-P). This enzyme is a member of the alpha-D-phosphohexomutase superfamily, which catalyzes the intramolecular phosphoryl transfer of sugar substrates. Here we report the crystal structures of AGM1 from Candida albicans for the first time, both in the apoform and in the complex forms with the substrate and the product, and discuss its catalytic mechanism. The structure of AGM1 consists of four domains, of which three domains have essentially the same fold. The overall structure is similar to those of phosphohexomutases; however, there are two additional beta-strands in domain 4, and a circular permutation occurs in domain 1. The catalytic cleft is formed by four loops from each domain. The N-acetyl group of the substrate is recognized by Val-370 and Asn-389 in domain 3, from which the substrate specificity arises. By comparing the substrate and product complexes, it is suggested that the substrate rotates about 180 degrees on the axis linking C-4 and the midpoint of the C-5-O-5 bond in the reaction.

O-Acetylation is a common decoration on endotoxins derived from many Gram-negative bacterial species, and it has been shown to be instrumental (e.g. in Salmonella typhimurium) in determining the final tertiary structure of the endotoxin and the immunogenicity of the molecule. Structural heterogeneity of endotoxins produced by mucosal pathogens such as Neisseria meningitidis is determined by decorations on the heptose inner core, including O-acetylation of the terminal N-acetylglucosamine (GlcNAc) attached to HepII. In this report, we show that O-acetylation of the meningococcal lipooligosaccharide (LOS) inner core has an important role in determining inner core assembly and immunotype expression. The gene encoding the LOS O-acetyltransferase, lot3, was identified by homology to NodX from Rhizobium leguminosarum. Inactivation of lot3 in strain NMB resulted in the loss of the O-acetyl group located at the C-3 position of the terminal GlcNAc of the LOS inner core. Inactivation of either lot3 or lgtG, which encodes the HepII glucosyltransferase, did not result in the appearance of the O-3-linked phosphoethanolamine (PEA) groups on the LOS inner core. Construction of a double mutant in which both lot3 and lgtG were inactivated resulted in the appearance of O-3-linked PEA groups on the LOS inner core. In conclusion, O-acetylation status of the terminal GlcNAc of the γ-chain of the meningococcal LOS inner core is an important determinant for the appearance or exclusion of the O-3-linked PEA group on the LOS inner core and contributes to LOS structural diversity. O-Acetylation also likely influences resistance to complement-mediated lysis and may be important in LOS conjugate vaccine design.

Lecanicillium fungicola was selected among 15 strains as chitinase producer and it was used to obtain the crude enzyme (UFL) in submerged fermentation (SF) with added chitin. The UFL displayed at pH 6 and 40 °C the highest endochitinase (Endo) and N-acetylhexosaminidase (NHase) activities, 747 and 410 U/mg, respectively. Four bands of proteins with molecular weights of 123.1, 85.5, 33.1 and 23 kDa were detected in UFL by SDS-PAGE. In order to increase solubilities of the substrates, α and β-chitins were treated with alkali; degrees of deacetylation (DD) were determined 55 and 50%, respectively. Thereafter, chitin hydrolysis with UFL was carried out at 40 °C and pH 5, Endo and NHase at these conditions were 619 and 355 U/mg of protein, respectively. The partial deacetylation as well as the use of acidified reaction media improved significantly the enzyme efficiency in terms of yields of chitin oligosaccharides produced and process time. The maximum chitin oligosaccharides concentration (Pmax) obtained from α- and β-deacetylated chitins were 2.77 and 4.44 mmol/l, respectively; whereas for α-chitin it was determined a very small amount of product (0.17 mmol/l). Despite of these results, the maximum production rate (Vmax 0.0836 mmol/l h) for α-deacetylated chitin was significantly higher than β-deacetylated chitin (0.0363 mmol/l h).

The N-acetyl-D-galactosamine (GalNAc) transport system of Escherichia coli K92 was studied when the bacterium was grown in a chemically defined medium containing GalNAc as a carbon source. Kinetic measurements were carried out in vivo at 37 degrees C in 25 mM phosphate buffer, pH 7.0. Under these conditions, the uptake rate was linear for at least 3 min and the calculated Km for GalNAc was 3 microM. The transport system was strongly inhibited by sodium arsenate (70%), potassium cyanide (62%) and 2,4-dinitrophenol (75%). Analysis of bacterial GalNAc phosphotransferase activity revealed in vitro GalNAc phosphorylation activity only when phosphoenolpyruvate was present. These results strongly support the notion that GalNAc uptake depends on a specific phosphotransferase system. Study of activity regulation showed that N-acetylglucosamine and mannosamine specifically inhibit the transport of GalNAc in this bacterium. Analysis of expression revealed that the GalNAc transport system is specifically induced by GalNAc but not by N-acetylglucosamine (GlcNAc) or N-acetylmannosamine (ManNAc), two intimately related sugars. Moreover, full induction of GalNAc transport required the presence of both cAMP and GalNAc. Comparative studies revealed that E. coli K92 has developed a regulation mechanism that specifically induces the appropriate permease based on the presence of each respective phospho-amino sugar (GlcNAc, ManNAc and GalNAc). In this regulation system, GlcNAc is the preferred amino sugar as the carbon source. Finally, when E. coli K92 was grown using GalNAc, capsular polysialic acid production was strongly affected. The presence of intracellular phosphoderivative acetylamino sugars, generated by the action of the phosphotransferase transport system, can be responsible for this effect.

The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain 10 polysaccharide deacetylase homologues. Six of these homologues have been proposed to be peptidoglycan N-acetylglucosamine deacetylases. Two of these genes, namely bc1960 and bc3618, have been cloned and expressed in Escherichia coli, and the recombinant enzymes have been purified to homogeneity and further characterized. Both enzymes were effective in deacetylating cell wall peptidoglycan from the Gram(+) Bacillus cereus and Bacillus subtilis and the Gram(–) Helicobacter pylori as well as soluble chitin substrates and N-acetylchitooligomers. However, the enzymes were not active on acetylated xylan. These results provide insight into the substrate specificity of carbohydrate esterase family 4 enzymes. It was revealed that both enzymes deacetylated only the GlcNAc residue of the synthetic muropeptide N-acetyl-d-glucosamine-(β-1,4)-N-acetylmuramyl-l-alanine-d-isoglutamine. Analysis of the constituent muropeptides of peptidoglycan from B. subtilis and H. pylori resulting from incubation of the enzymes BC1960 and BC3618 with these polymers and subsequent hydrolysis by Cellosyl and mutanolysin, respectively, similarly revealed that both enzymes deacetylate GlcNAc residues of peptidoglycan. Kinetic analysis toward GlcNAc2–6 revealed that GlcNAc4 was the favorable substrate for both enzymes. Identification of the sequence of N-acetychitooligosaccharides (GlcNAc2–4) following enzymatic deacetylation by using 1H NMR revealed that both enzymes deacetylate all GlcNAc residues of the oligomers except the reducing end ones. Enzymatic deacetylation of chemically acetylated vegetative peptidoglycan from B. cereus by BC1960 and BC3618 resulted in increased resistance to lysozyme digestion. This is the first biochemical study of bacterial peptidoglycan N-acetylglucosamine deacetylases.

The adhesion of the pathogen Neisseria meningitidis to host cell surface proteoglycan, mediated by the integral outer membrane proteins OpcA and Opa, plays an important part in the processes of colonization and invasion by the bacterium. The precise specificities of the OpcA and Opa proteins are, however, unknown. Here we use a fluorescence-based binding assay to show that both proteins bind to mono- and disaccharides with high affinity. Binding of saccharides caused a quench in the intrinsic fluorescence emission of both proteins, and mutation of selected Tyr residues within the external loop regions caused a substantial decrease in fluorescence. We suggest that the intrinsic fluorescence arises from resonance energy transfer from Tyr to Trp residues in the β-barrel portion of the structure. OpcA bound sialic acid with a Kd of 0.31 μm and was shown to be specific for pyranose saccharides. The binding specificities of two different Opa proteins were compared; unlike OpcA, neither protein bound to monosaccharides, but both bound to maltose, lactose, and sialic acid-containing oligosaccharides, with Kd values in the micromolar range. OpaB had a 10-fold higher affinity for sialic acid-containing ligands than OpaD as a result of the mutation Y165V, which was shown to restore this specificity to OpaD. Finally, the OpcA- and Opa-dependent adhesion of meningococci to epithelial cells was shown to be partially inhibited by exogenously added sialic acid and maltose. The results show that OpcA and the Opa proteins can be thought of as outer membrane lectins and that simple saccharides can modulate their recognition of complex proteoglycan receptors.

A method for meningococcal X (MenX) polysaccharide quantification by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) is described. The polysaccharide is hydrolyzed by strong acidic treatment, and the peak of glucosamine-4-phosphate (4P-GlcN) is detected and measured after chromatography. In the selected conditions of hydrolysis, 4P-GlcN is the prevalent species formed, with GlcN detected for less than 5% in moles. As standard for the analysis, the monomeric unit of MenX polysaccharide, N-acetylglucosamine-4-phosphate (4P-GlcNAc), was used. This method for MenX quantification is highly selective and sensitive, and it constitutes an important analytical tool for the development of a conjugate vaccine against MenX.