Human Endogenous Metabolite

There is increasing evidence that in several fungi, rhamnose-containing glycans are involved in processes that affect host-pathogen interactions, including adhesion, recognition, virulence, and biofilm formation. Nevertheless, little is known about the pathways for the synthesis of these glycans. We show that rhamnose is present in glycans isolated from the rice pathogen Magnaporthe grisea and from the plant pathogen Botryotinia fuckeliana. We also provide evidence that these fungi produce UDP-rhamnose. This is in contrast to bacteria where dTDP-rhamnose is the activated form of this sugar. In bacteria, formation of dTDP-rhamnose requires three enzymes. Here, we demonstrate that in fungi only two genes are required for UDP-Rha synthesis. The first gene encodes a UDP-glucose-4,6-dehydratase that converts UDP-glucose to UDP-4-keto-6-deoxyglucose. The product was shown by time-resolved (1)H NMR spectroscopy to exist in solution predominantly as a hydrated form along with minor amounts of a keto form. The second gene encodes a bifunctional UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase that converts UDP-4-keto-6-deoxyglucose to UDP-rhamnose. Sugar composition analysis and gene expression studies at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under tissue-specific regulation. Together, our results provide new insight into the formation of rhamnose-containing glycans during the fungal life cycle. The role of these glycans in the interactions between fungal pathogens and their hosts is discussed. Knowledge of the metabolic pathways involved in the formation of rhamnose-containing glycans may facilitate the development of drugs to combat fungal diseases in humans, as to the best of our knowledge mammals do not make these types of glycans[1].

UG4,6-Dh and U4k6dG-ER Enzyme Assays [1]
UG4,6-Dh reactions (50-μl final volume) were carried out in 50 mm sodium phosphate, pH 7.6, containing 1 mm NAD+, 2 mm UDP-Glc, and 13 μg of purified recombinant UG4,6-Dh. U4k6dG-ER reactions (60-μl final volume) were carried out in 50 mm sodium phosphate, pH 7.6, containing 3 mm NADPH, 1 mm NAD+, 2 mm UDP-Glc, purified recombinant UG4,6-Dh, and purified recombinant U4k6dG-ER (17 μg). Reactions were kept for up to 45 min at 30 °C and then terminated (100 °C water bath, 1 min). Chloroform (50 μl) was added; the mixture was vortexed and centrifuged (12,000 rpm, 5 min at 22 °C). The upper aqueous phase was collected. Reaction products were chromatographed on an anion-exchange column (Q15 resin in 1-mm inner diameter × 250 mm; or Mono Q 5 × 50 mm) eluted with a linear gradient of ammonium formate (5 mm to 0.6 m) over 25 min, using an Agilent 1200 Series HPLC system equipped with an autosampler, diode array detector, and ChemStation software. Nucleotides were detected by their A261 (maximum for UDP-sugar) and A259 (maximum for NAD+). The amount of product formed was determined using a calibration curve of standard UDP-Glc. The products formed by the UG4,6-Dh reaction (eluted at 12.3 min from Q-column) and the U4k6dG-ER reaction (eluted at 13.2 min from the PA1 column) were collected, lyophilized, dissolved in water or 99.9% D2O, and analyzed by ESI-MS and by 1H NMR spectroscopy.

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Real Time 1H NMR-based Enzyme Assay [1]
Real time 1H NMR spectroscopic monitoring of enzymatic reactions (150 μl) was performed at 30 °C in a mixture of D2O/H2O (9:1 v/v), containing 50 mm sodium phosphate at various pH values, and 1–1.5 mm UDP-Glc, 0.6 mm NAD+, and 13 μg of recombinant UG4,6-Dh. The combined UG4,6-Dh and U4k6dG-ER NMR assay used the same buffer supplemented with 1.5 mm NADPH. Real time 1H NMR spectra were obtained using a Varian VNMRS spectrometer operating at 600 MHz and equipped with a 3-mm cold probe. Data acquisition was started between 2 and 5 min after the addition of enzyme to allow spectrometer acquisition conditions to be optimized. Sequential one-dimensional proton spectra with presaturation of the water resonance were acquired over the course of the enzymatic reaction, with spectra summed and averaged every 8 s. All chemical shifts (ppm) are referenced to 2,2-dimethyl-2-silapentane-5-sulfonate (δ0.00). 13C-Enriched UDP-4-keto-6-deoxyglucose was prepared from UDP-[13C]Glc using recombinant Sloppy (UDP-sugar pyrophosphorylase (USP)), UTP, and [13C]Glc-1-P. Subsequently, the 4,6-dehydratase with or without the U4k6dG-ER was added. The reaction mixtures, which contained UDP-[13C]Glc, UDP-[13C]4k6dG, and UDP-[13C]Rha, were examined by 13C HSQC and 13C HMBC NMR experiments using standard Varian pulse programs.

[1]. Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana. J Biol Chem. 2012 Jan 6;287(2):879-92.

UDP-beta-L-rhamnose is a UDP-L-rhamnose in which the rhamnose portion has beta-configuration at its anomeric centre
Udp-beta-L-rhamnose has been reported in Arabidopsis thaliana with data available.

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Based on genomic DNA sequence data, fungal species that have Rha-containing glycans also carry genes involved in UDP-4-keto-6-deoxyglucose and UDP-rhamnose synthesis. For example, S. schenckii, Candida, C. graminicola, and T. melanosporum have genes with homology to the M. grisea and B. fuckeliana genes identified in this report (as shown in supplemental Figs. S3B and S6B). Interestingly, the UDP-rhamnose biosynthetic genes of plant fungi are closer by phylogeny analysis to the plant species, when compared with the animal fungal biosynthetic genes. Of note is the clustering of the two viral UGlc4,6-Dh proteins. The chlorella virus co-living with plant appears to cluster with UGlc4,6-Dh in plants, although the same protein from the giant DNA virus that infects members of the genus Acanthamoeba is clustered with Trypanosoma (supplemental Fig. S3B). It would be of interest in the future to look at many viral genomes and determine the role of UDP-Rha in these virus-host interactions. As many as 200 fungi are known to impact human health. As far as we are aware, humans do not make Rha-containing glycans; thus, the metabolic pathways involved in the formation of rhamnose-containing glycans provide a potential target for drugs to control some fungal diseases in humans as well as other animals. Additional research is required to structurally characterize the Rha-containing glycans synthesized by M. grisea and B. fuckeliana. Such information together with data obtained after reduction or elimination of these two UDP-Rha biosynthetic genes, will further illuminate the roles of rhamnose-containing glycans in fungi.[1]

C15H24N2O16P2

550.30

550.06

1955-26-6

192751

White to off-white solid powder

1.9g/cm3

1.652

-6.1

8

16

8

35

948

9

C[C@H]1[C@@H]([C@H]([C@H]([C@H](O1)OP(=O)(O)OP(=O)(O)OC[C@@H]2[C@H]([C@H]([C@@H](O2)N3C=CC(=O)NC3=O)O)O)O)O)O

DRDCJEIZVLVWNC-SLBWPEPYSA-N

InChI=1S/C15H24N2O16P2/c1-5-8(19)10(21)12(23)14(30-5)32-35(27,28)33-34(25,26)29-4-6-9(20)11(22)13(31-6)17-3-2-7(18)16-15(17)24/h2-3,5-6,8-14,19-23H,4H2,1H3,(H,25,26)(H,27,28)(H,16,18,24)/t5-,6+,8-,9+,10+,11+,12+,13+,14+/m0/s1

[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl] hydrogen phosphate

Udp-beta-L-rhamnose; 1955-26-6; UDP-rhamnose; UDP-L-rhamnose; [[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl] hydrogen phosphate; CHEBI:84725; Uridine-5'-Monophosphate Glucopyranosyl-Monophosphateester; uridine 5'-[3-(beta-L-rhamnopyranosyl) dihydrogen diphosphate];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 10 mg/mL (18.17 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)