Diverse Sugar Research Articles

Achievements and impacts of glycosylation reactions involved in natural product biosynthesis in prokaryotes

Bioactive natural products, such as polyketides, flavonoids, glycopeptides, and aminoglycosides, have been used as therapeutic agents. Many of them contain structurally diverse sugar moieties attached to the aglycone core structures. Glycosyltransferases (GTs) catalyze the attachment of nucleotide-activated sugar substrates to acceptor aglycones. Because these sugar moieties are usually essential for biological activity, in vivo pathway engineering in prokaryotic hosts and in vitro enzymatic approaches coupled with GT engineering are currently being used to synthesize novel glycosylated derivatives, and some of them exhibited improved biological activities compared to the parent molecules. Therefore, harnessing the potential of diverse glycosylation reactions in prokaryotes will increase the structural diversity of natural products and the possibility to generate new bioactive products.

Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP)

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.

Pathways leading to 3-amino- and 3-nitro-2,3-dideoxy sugars: strategies and synthesis

3-Amino- and 3-nitro-2,3-dideoxy sugars, which are structurally diverse and uncommon sugars bearing an amino substitution on a sugar scaffold, have been shown to play crucial roles in the biological activities of aminoglycoside antibiotics and aminoglycoside containing natural products. Over the past few decades, there have been continuing efforts on the convenient synthesis of monosaccharides and oligosaccharides containing 3-amino- and 3-nitro-2,3-dideoxysugar motifs, beginning from either carbohydrate or non-carbohydrate precursors. This review article is intended to provide an updated overview of the most striking contributions in this field, based on the various strategies to construct the 3-amino sugar ring.

Agrobacterium tumefaciens recognizes its host environment using ChvE to bind diverse plant sugars as virulence signals

Agrobacterium tumefaciens is a broad host range plant pathogen that combinatorially recognizes diverse host molecules including phenolics, low pH, and aldose monosaccharides to activate its pathogenic pathways. Chromosomal virulence gene E (chvE) encodes a periplasmic-binding protein that binds several neutral sugars and sugar acids, and subsequently interacts with the VirA/VirG regulatory system to stimulate virulence (vir) gene expression. Here, a combination of genetics, X-ray crystallography, and isothermal calorimetry reveals how ChvE binds the different monosaccharides and also shows that binding of sugar acids is pH dependent. Moreover, the potency of a sugar for vir gene expression is modulated by a transport system that also relies on ChvE. These two circuits tune the overall system to respond to sugar concentrations encountered in vivo. Finally, using chvE mutants with restricted sugar specificities, we show that there is host variation in regard to the types of sugars that are limiting for vir induction.

Diversity and Versatility of the Thermotoga maritima Sugar Kinome

Sugar phosphorylation is an indispensable committed step in a large variety of sugar catabolic pathways, which are major suppliers of carbon and energy in heterotrophic species. Specialized sugar kinases that are indispensable for most of these pathways can be utilized as signature enzymes for the reconstruction of carbohydrate utilization machinery from microbial genomic and metagenomic data. Sugar kinases occur in several structurally distinct families with various partially overlapping as well as yet unknown substrate specificities that often cannot be accurately assigned by homology-based techniques. A subsystems-based metabolic reconstruction combined with the analysis of genome context and followed by experimental testing of predicted gene functions is a powerful approach of functional gene annotation. Here we applied this integrated approach for functional mapping of all sugar kinases constituting an extensive and diverse sugar kinome in the thermophilic bacterium Thermotoga maritima. Substrate preferences of 14 kinases mainly from the FGGY and PfkB families were inferred by bioinformatics analysis and biochemically characterized by screening with a panel of 45 different carbohydrates. Most of the analyzed enzymes displayed narrow substrate preferences corresponding to their predicted physiological roles in their respective catabolic pathways. The observed consistency supports the choice of kinases as signature enzymes for genomics-based identification and reconstruction of sugar utilization pathways. Use of the integrated genomic and experimental approach greatly speeds up the identification of the biochemical function of unknown proteins and improves the quality of reconstructed pathways.

Evolutionary Divergence of Sedoheptulose 7-Phosphate Cyclases Leads to Several Distinct Cyclic Products

Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases, and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3-1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules.

Selective derivatization of nucleotide diphosphate (NDP)-4-keto sugars for electrospray ionization-mass spectrometry (ESI-MS)

Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process. In this study, we evaluated various hydroxylamine compounds for the derivatization of NDP-4-keto sugars, so that the detection of the sugars by ESI-MS is more efficient. As a result, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine was found to be the most effective tagging molecule for the detection of NDP-4-keto sugars without being interfered by original MS. This method can be used for identifying NDP-4-keto sugars such as thymidine diphosphate (TDP)-, adenosine diphosphate (ADP)-, uridine diphosphate (UDP)-, and cytosine diphosphate (CDP)-4-keto sugars as well as new NDP-4-keto-dehydratases.

Structural Studies of the Spinosyn Rhamnosyltransferase, SpnG

Spinosyns A and D (spinosad), like many other complex polyketides, are tailored near the end of their biosyntheses through the addition of sugars. SpnG, which catalyzes their 9-OH rhamnosylation, is also capable of adding other monosaccharides to the spinosyn aglycone (AGL) from TDP-sugars; however, the substitution of UDP-D-glucose for TDP-D-glucose as the donor substrate is known to result in a >60000-fold reduction in k(cat). Here, we report the structure of SpnG at 1.65 Å resolution, SpnG bound to TDP at 1.86 Å resolution, and SpnG bound to AGL at 1.70 Å resolution. The SpnG-TDP complex reveals how SpnG employs N202 to discriminate between TDP- and UDP-sugars. A conformational change of several residues in the active site is promoted by the binding of TDP. The SpnG-AGL complex shows that the binding of AGL is mediated via hydrophobic interactions and that H13, the potential catalytic base, is within 3 Å of the nucleophilic 9-OH group of AGL. A model for the Michaelis complex was constructed to reveal the features that allow SpnG to transfer diverse sugars; it also revealed that the rhamnosyl moiety is in a skew-boat conformation during the transfer reaction.

Glycoconjugates Play a Key Role in Campylobacter jejuni Infection: Interactions between Host and Pathogen

Glycan based interactions between host and pathogen are critical in many bacterial and viral diseases. Glycan interactions range from initial receptor based adherence to protecting the infective agent from the host’s immune response through molecular mimicry. Campylobacter jejuni is an ideal model for studying the role of glycans in host–pathogen interactions, as well as the role of bacterial surface glycoconjugates in infection. Using glycan array analysis, C. jejuni has been shown to interact with a wide range of host glycoconjugates. Mannose and sialic acid residues appear to play a role in initial interactions between host and pathogen following environmental exposure, whereas fucose and galactose based interactions are likely to be required for prolonged colonization. Other studies have highlighted potential decoy receptor type interactions between host’s intestinal mucins and C. jejuni, demonstrating the importance of host glycoproteins as defense against C. jejuni infection as well as the role for glycoconjugates found in human breast milk in protection of breast feeding infants from infection with C. jejuni. C. jejuni can produce N- and O-linked glycoproteins, capsular polysaccharide (CPS) and/or lipooligosaccharide (LOS) which results in C. jejuni presenting its own diverse sugar coated displays on the cell surface. Bacterial glycans play an important and versatile role in infection and disease. Of these, the best understood is the molecular mimicry of human gangliosides presented by C. jejuni’s LOS and its link to the onset of autoimmune neuropathies such as the Guillain Barrè syndrome (GBS). However, the role of glycoconjugates presented by C. jejuni extends beyond expression of sialylated ganglioside structures involved in initiation of GBS. Expression of surface glycans by C. jejuni may also relate to the ability of this organism to interact with the glycoproteins for initial host–pathogen interactions and continued infectivity.

Characterization of Methylobacterium strains isolated from the phyllosphere and description of Methylobacterium longum sp. nov

Methylobacterium strains are abundantly found in the phyllosphere of plants. Morphological, physiological and chemotaxonomical properties of 12 previously isolated strains were analyzed in order to obtain a more detailed overview of the characteristics of phyllosphere colonizing Methylobacterium strains. All strains showed the typical properties of the genus Methylobacterium, including pink pigmentation, facultative methylotrophy, a fatty acid profile dominated by C18:1 ω7c, and a high G+C content of 65mol % or more. However, some strains showed only weak growth on methanol and pigmentation varied from pale pink to red. Strains grew best under mesophilic, neutrophilic conditions and low salt (≤1%) concentrations, but variation was seen with respect to the temperature and pH range under which growth occurred. Likewise, differences were seen with respect to carbon source utilization. Some strains were versatile and utilized diverse organic acids, amino acids and sugars, while others could only metabolize a restricted number of organic acids. The strains that were most distinct from existing type strains based on 16S rRNA gene sequence analysis were selected for DNA-DNA hybridization experiments to analyze whether they are sufficiently different at the genomic level from existing type strains to justify their classification as new species. This resulted in the delineation of strain 440 and its description as Methylobacterium longum sp. nov. strain 440 (=DSM 23933(T)=CECT 7806(T)). A main characteristic of this species is the formation of relatively long rods compared to other Methylobacterium species.

Engineered biosynthesis of glycosylated derivatives of narbomycin and evaluation of their antibacterial activities

A 14-membered macrolide antibiotic narbomycin produced from Streptomyces venezuelae ATCC 15439 is composed of polyketide macrolactone ring and D-desosamine as a deoxysugar moiety, which acts as an important determinant of its antibacterial activity. In order to generate diverse glycosylated derivatives of narbomycin, expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into S. venezuelae YJ003 mutant strain bearing a deletion of thymidine-5'-diphospho-D-desosamine biosynthetic gene cluster. The resulting recombinants of S. venezuelae produced a range of new analogs of narbomycin, which possess unnatural sugar moieties instead of native deoxysugar D-desosamine. The structures of narbomycin derivatives were determined through nuclear magnetic resonance spectroscopy and mass spectrometry analyses and their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose or 3-O-demethyl-D-chalcose was demonstrated to exhibit greater antibacterial activity than narbomycin and the clinically relevant erythromycin. This work provides new insight into the functions of deoxysugar biosynthetic enzymes and structure-activity relationships of the sugar moieties attached to the macrolides and demonstrate the potential of combinatorial biosynthesis for the generation of new macrolides carrying diverse sugars with increased antibacterial activities.

Ceric Ammonium Nitrate-Catalyzed Azidation of 1,2-Anhydro Sugars: Application in the Synthesis of Structurally Diverse Sugar-Derived Morpholine 1,2,3-Triazoles and 1,4-Oxazin-2-ones

Azidation of 1,2-anhydro sugars with NaN(3) in CH(3)CN by using a catalytic amount of ceric ammonium nitrate has been accomplished in a regio- and stereoselective manner. Various 1,2-anhydro sugars produced 2-hydroxy-1-azido sugars in good yields which, in turn, were converted to structurally diverse sugar-derived morpholine triazoles and sugar oxazin-2-ones. These sugar derivatives were tested against various commercially available glycosidases, and two of them were found to be active in the micromolar range.

Warfarin Glycosylation Invokes a Switch from Anticoagulant to Anticancer Activity

Warfarin Glycosylation Invokes a Switch from Anticoagulant to Anticancer Activity

Facile fabrication of promising protein tyrosine phosphatase (PTP) inhibitor entities based on ‘clicked’ serine/threonine–monosaccharide hybrids

Protein tyrosine phosphatases (PTPs) are well-validated therapeutic targets for many human major diseases. The development of their potent inhibitors has therefore become a main focus of both academia and the pharmaceutical industry. We report herein a facile strategy toward the fabrication of new and competent PTP inhibitor entities by simply ’clicking’ alkynyl amino acids onto diverse azido sugar templates. Triazolyl glucosyl, galactosyl, and mannosyl serine and threonine derivatives were efficiently synthesized via click reaction, which were then identified as potent CDC25B and PTP1B inhibitors selective over a panel of homologous PTPs tested. Their inhibitory activity and selectivity were found to largely lie on the structurally and configurationally diversified monosaccharide moieties whereon serinyl and threoninyl residues were introduced. In addition, MTT assay revealed the triazole-connected sugar-amino acid hybrids may also inhibit the growth of several human cancer cell lines including A549, Hela, and especially HCT-116. On the basis of such compelling evidence, we consider that this compound series could furnish promising chemical entities serving as new CDC25B and PTP1B inhibitors with potential cellular activity. Furthermore, the ‘click’ strategy starting from easily accessible and biocompatible amino acids and sugar templates would allow the modular fabrication of a rich library of new PTP inhibitors efficaciously and productively.

미생물 유래 특이당을 이용한 플라보노이드 당화반응

Glycosylation is a key mechanism in determining diversity of natural products, and influencing their bioactivities. This approach requires a core set of glycosyltransferase that synthesizes the diverse sugar structures observed in nature. Recently, the researchers have begun to alter the sugar moiety and glycosylation patterns of natural products both in vivo E. coli system and in vitro for their glycodiversification. This review highlights new glycosylation tools using microbialproduced deoxysugar and a flexible glycosyltransferase on natural plant-flavonoids to generate novel glycoforms with useful biological activity.

Production of novel platycodin D derivatives by Aspergilli

Platycodin D (PD) is the major saponin components abundant in the root of Platycodon grandiflorum, which has been reported to have anti‐inflammatory and anti‐tumor activities. PD contains sugar residues including glucose unit at C‐3 and apiose‐xylose‐rhamnose‐arabinose residue at C‐28. In the present study, 8 noble PD derivatives with diverse sugar chains were produced by the crude enzyme extracts of Aspergillus niger and A. usamii grown in the presence of various carbon sources. Commercially available enzymes such as hesperidinase, β‐glucosidase, and β‐xylosidase were not effective in cleaving the glycosidic side chains of PD. Depending on the sugar side chains the PD derivatives demonstrated different degree of toxicity against a normal fetal colon cell line. These findings could be informative to obtain novel PD derivatives with reduced cytotoxicity by Aspergilli. This work was supported by Industrialization Support Program for Bio‐technology of Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (810007‐03‐1‐WT211), Republic of Korea.

Combining Agriculture with Microbial Genomics To Make Fuels

Despite major challenges, we believe that cellulosic sugars in agricultural waste are the best sustainable source for biofuels to meet world wide energy needs. One key challenge involves dealing with diverse cellulosic sugars, many with high levels of lignin. Other tough challenges include converting both 5-and 6-carbon sugars efficiently into ethanol and recycling carbon dioxide from fermentations back into useful biomass. We would like to develop “integrated biorefineries” that will use residual biomass from conventional crops and also harness microbial genomics to identify enzymes with which to process biomass produced in marginal lands with much greater efficiency than is now possible.

Advanced Glycation End Products Subspecies-Selectively Induce Adhesion Molecule Expression and Cytokine Production in Human Peripheral Blood Mononuclear Cells

Advanced glycation end products (AGEs) are proteins or lipids that become glycated after exposure to diverse reducing sugars. Accumulation of AGEs induces diabetes complications. Microinflammation is a common major mechanism in the pathogenesis of diabetic vascular complications. Activation of monocytes/macrophages and T cells plays roles in the pathogenesis of atherosclerosis. The activation of T cells requires the enhanced expression of adhesion molecules on monocytes. AGEs activate monocytes by engaging the receptor for AGE (RAGE); however, little is known about the profile of agonist activity of diverse AGE moieties on monocytes. We investigated the effect of four distinct AGE subtypes (AGE-modified bovine serum albumin; AGE-2, AGE-3, AGE-4, and AGE-5) at concentrations ranging from 0.1 to 100 microg/ml on the expression of intercellular adhesion molecule-1, B7.1, B7.2, and CD40 on monocytes and its impact on the production of interferon-gamma and tumor necrosis factor-alpha in human peripheral blood mononuclear cells. Among the AGEs examined, AGE-2 and AGE-3 selectively induced adhesion molecule expression and cytokine production. Antagonism experiments using antibodies against adhesion molecules demonstrated that cell-to-cell interaction between monocytes and T/natural killer cells was involved in AGE-2- and AGE-3-induced cytokine production. AGE-2 and AGE-3 up-regulated the expression of RAGE on monocytes. The effects of AGE-2 and AGE-3 were inhibited by nuclear factor-kappaB and p38 mitogen-activated protein kinase inhibitors. These results indicated that AGE-2 and AGE-3 activated monocytes via RAGE, leading to the up-regulation of adhesion molecule expression and cytokine production.

Natural-product sugar biosynthesis and enzymatic glycodiversification.

Many biologically active small-molecule natural products produced by microorganisms derive their activities from sugar substituents. Changing the structures of these sugars can have a profound impact on the biological properties of the parent compounds. This realization has inspired attempts to derivatize the sugar moieties of these natural products through exploitation of the sugar biosynthetic machinery. This approach requires an understanding of the biosynthetic pathway of each target sugar and detailed mechanistic knowledge of the key enzymes. Scientists have begun to unravel the biosynthetic logic behind the assembly of many glycosylated natural products and have found that a core set of enzyme activities is mixed and matched to synthesize the diverse sugar structures observed in nature. Remarkably, many of these sugar biosynthetic enzymes and glycosyltransferases also exhibit relaxed substrate specificity. The promiscuity of these enzymes has prompted efforts to modify the sugar structures and alter the glycosylation patterns of natural products through metabolic pathway engineering and enzymatic glycodiversification. In applied biomedical research, these studies will enable the development of new glycosylation tools and generate novel glycoforms of secondary metabolites with useful biological activity.

Carbohydrate-Metal Complexes and their Potential as Anticancer Agents

Platinum complex-based chemotherapy is one of the major treatment options of many malignancies. Although severe side effects occur, and only a limited spectrum of tumors can be cured, Pt compounds are used in every second therapy scheme. Thus, many different drug design strategies have been employed for improving the properties of anticancer drugs including pH or redox activation in the tumor, variation of the metal center and therefore the redox and ligand exchange properties, the application of multinuclear metal complexes, the development of targeted approaches, etc. Application of carbohydrate-metal complexes is an example of a targeted approach exploiting the biochemical and metabolic functions of diverse sugars in living organisms for transport and accumulation. Natural carbohydrates and synthetic derivatives possess a manifold of donors endowing them with the ability to coordinate metal centers and providing some additional advantages over other ligands, e.g., biocompatibility, non-toxicity, enantiomeric purity, water solubility, and well-explored chemistry. In recent years, several examples of carbohydrate compounds have been developed for diverse medicinal applications ranging from compounds with antibiotic, antiviral, or fungicidal activity and anticancer compounds. Herein, metal complexes with carbohydrate ligands are reviewed and the role of the carbohydrate carriers on the antineoplastic activity of these compounds, both in vitro and in vivo, is described.