UDP-glucose 4-epimerase Activity Research Articles

Study of the Differentially Expressed Genes in the Pomacea canaliculata Transcriptome after Treatment with Pedunsaponin A.

Transcriptomes, genomes, and proteomes have played important roles in the search for drug targets. To determine the molluscicidal mechanism of pedunsaponin A against Pomacea canaliculata, RNA-seq technology was adopted to analyze the differentially expressed genes (DEGs) in the P. canaliculata transcriptome after treatment with pedunsaponin A. As a result, 533 DEGs were identified, among which 255 genes were significantly upregulated and 278 genes were significantly downregulated. According to the analysis of Gene Ontology (GO) functions, we found that the DEGs were significantly enriched in the viral life cycle, UDP-glucose 4-epimerase activity, guanylate cyclase activity, the cyclic guanosine monophosphate (cGMP) biosynthetic process, and the cGMP metabolic process. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway results showed that the DEGs were mainly involved in the hedgehog signaling pathway, phagosome, cytosolic DNA-sensing pathway, retinoic acid-inducible gene I like (RIG-I-like) receptor signaling pathway, bacterial secretion system, and nuclear factor-kappa B (NF-kappa B) signaling pathway. The above results indicated that pedunsaponin A causes a metabolic disorder, anomalous opening of membrane ion channels, and an imbalance in osmotic pressure between the interior and exterior of cells, eventually resulting in the death of cells involved in immune defense and influencing the immune response of P. canaliculata.

Molecular cloning and functional analysis of DoUGE related to water-soluble polysaccharides from Dendrobium officinale with enhanced abiotic stress tolerance

UDP glucose 4-epimerase (UGE), an enzyme with significant impacts on sugar metabolism, catalyzes the reversible inter-conversion between UDP-glucose and UDP-galactose. However, very little is known about whether UGE plays a critical role in the accumulation of water-soluble polysaccharide (WSP) and its relationship to abiotic stress tolerance. Here, DoUGE from D. officinale, encoding UGE localized in the cytoplasm, was initially cloned and analyzed. DoUGE exhibited highly tissue-specific expression patterns. The highest expression was in the stems of seedlings and adult plants. The content of WSPs ranged from 168.43 to 416.12 mg g−1 DW from developmental stages S1 to S4, the highest value being in S3. DoUGE was expressed throughout S1 to S4, with a maximum in S3. This trend was similar in three cultivated varieties (T10, T32-5 and T636). There was a positive correlation between DoUGE expression and the content of WSPs (R 2 = 0.94; p < 0.01). Furthermore, promoter analysis showed its possible role in responses to abiotic stresses. Transgenic Arabidopsis thaliana seedlings overexpressing DoUGE accumulated 34.84–44.78% more WSPs, showed 26.24–32.79% more UGE activity, and had a 1.19–1.31-fold higher chlorophyll content than the wild type. Transgenic plants also showed a 50.84 and 34.33% increase in the average content of glucose and galactose, respectively. Transgenic lines growing in half-strength Murashige and Skoog medium containing 150 mM NaCl or 200 mM mannitol displayed enhanced root length and fresh weight, as well as lower proline and malondialdehyde accumulation under salt and osmotic stresses, indicating that the DoUGE gene could be used to improve tolerance to abiotic stress in crops and medicinal or ornamental plants. Our results provide genetic evidence for the involvement of DoUGE in the regulation of WSP content during plant development in D. officinale, as well as in enhanced tolerance to salt and osmotic stresses.

Bifunctional cytosolic UDP-glucose 4-epimerases catalyse the interconversion between UDP-D-xylose and UDP-L-arabinose in plants.

UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The N-terminal amino acid sequence of the enzyme revealed that it is encoded by a predicted UDP-Glc 4-epimerase gene, PsUGE1, and is distinct from the UDP-Xyl 4-epimerase localized in the Golgi apparatus. rPsUGE1 (recombinant P. sativum UGE1) expressed in Escherichia coli exhibited both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities with apparent Km values of 0.31, 0.29, 0.16 and 0.15 mM for UDP-Glc, UDP-Gal (UDP-galactose), UDP-Ara (UDP-L-arabinose) and UDP-Xyl respectively. The apparent equilibrium constant for UDP-Ara formation from UDP-Xyl was 0.89, whereas that for UDP-Gal formation from UDP-Glc was 0.24. Phylogenetic analysis revealed that PsUGE1 forms a group with Arabidopsis UDP-Glc 4-epimerases, AtUGE1 and AtUGE3, apart from a group including AtUGE2, AtUGE4 and AtUGE5. Similar to rPsUGE1, recombinant AtUGE1 and AtUGE3 expressed in E. coli showed high UDP-Xyl 4-epimerase activity in addition to their UDP-Glc 4-epimerase activity. Our results suggest that PsUGE1 and its close homologues catalyse the interconversion between UDP-Xyl and UDP-Ara as the last step in the cytosolic de novo pathway for UDP-Ara generation. Alternatively, the net flux of metabolites may be from UDP-Ara to UDP-Xyl as part of the salvage pathway for Ara.

UDP-sugar biosynthetic pathway: contribution to cyanidin 3-galactoside biosynthesis in apple skin

UDP-galactose:flavonoid 3-O-galactosyltransferase (UFGalT) is responsible for cyanidin 3-galactoside (cy3-gal) synthesis from cyanidin (cy) and UDP-galactose (UDP-gal) which are, respectively, catalyzed by anthocyanidin synthase (ANS) and UDP-glucose 4-epimerase (UGE). To clarify the contribution of UDP-galactose pathway to cy3-gal accumulation in apple skin, we analyzed the contents of UDP-gal and UDP-glucose (UDP-glu), cy, and, cy3-gal contents along with UGE activity. We confirmed that transcript levels for apple ANS and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) coincided with anthocyanin accumulation in three apple cultivars differing in their skin colors. During fruit development, changes in level of cy coincided with that of cy3-gal, whereas UDP-gal and UGE activity showed no similar trend with cy3-gal. Significant correlation was not observed between the changes in UGE activity and UDP-sugar contents. The effect of temperature and UV-B radiation (different environmental conditions) on the accumulation of UDP-sugars, cy and cy3-gal, and UGE activity were also investigated in a pale-red cultivar. High temperature tended to depress the accumulation of both UDP-sugars and cy concomitant with the decrease in cy3-gal content irrespective of UV-B radiation. Although there was no high inhibition of both cy and UDP-sugars at low-temperature without UV-B, cy3-gal accumulation was highly depressed. UGE activity was highest at low temperature with UV-B, but not much different under other conditions. Most of the parameters under different environmental conditions were significantly correlated with each other. Based on these results, contribution of UDP-sugar biosynthetic pathway to anthocyanin biosynthesis under different environmental conditions as well as during fruit development is discussed.

UDP-Glucose 4-Epimerase Isoforms UGE2 and UGE4 Cooperate in Providing UDP-Galactose for Cell Wall Biosynthesis and Growth of Arabidopsis thaliana

Five Arabidopsis thaliana genes that encode UDP-glucose 4-epimerase (UGE) and represent two ancient plant UGE clades might be involved in the regulation of cell wall carbohydrate biosynthesis. We tested this hypothesis in a genome-wide reverse genetic study. Despite significant contributions of each gene to total UGE activity, none was essential for normal growth on soil. uge2 uge4 displayed dramatic general growth defects, while other mutant combinations were partially aberrant. UGE2 together with UGE3 influenced pollen development. UGE2 and UGE4 synergistically influenced cell wall galactose content, which was correlated with shoot growth. UGE2 strongly and UGE1 and UGE5 lightly supported UGE4 in influencing root growth and cell wall galactose content by affecting galactan content. By contrast, only UGE4 influenced xyloglucan galactosylation in roots. Secondary hypocotyl thickening and arabinogalactan protein carbohydrate structure in xylem parenchyma depended on the combination of UGE2 and UGE4. As opposed to cell wall galactose content, tolerance to external galactose strictly paralleled total UGE activity. We suggest a gradual recruitment of individual UGE isoforms into specific roles. UGE2 and UGE4 influence growth and cell wall carbohydrate biosynthesis throughout the plant, UGE3 is specialized for pollen development, and UGE1 and UGE5 might act in stress situations.

Characterization of RP 333, a gene encoding CapD of Rickettsia prowazekii with UDP-glucose 4-epimerase activity

The genetic mechanism of membrane assembly in Rickettsia prowazekii, the causative agent of epidemic typhus, is unknown. Recent progress in genomics of this small intracellular bacterium prompted us to explore membrane biogenesis as it pertains to identification and characterization of gene products that are of importance in polysaccharide biosynthesis. We chose to define the function of the R. prowazekii capD gene (RP 333) product designated as a putative epimerase. We detected a capD transcript in yolk sacs infected with R. prowazekii at ten days post-infection. The capD gene was PCR amplified and cloned into the entry vector pENTR/SD/D-TOPO and transferred to a low copy arabinose-inducible pBAD/ Myc-HisA expression plasmid constructed in our laboratory. The corresponding recombinant protein (CapD) was expressed in PL2, a galE28 mutant of Escherichia coli, and has been shown to epimerize UDP-galactose to UDP-glucose without the addition of NAD +. CapD characterization is important for defense strategies targeted towards design of an R. prowazekii vaccine, generation of new antibiotics, and opening the way for research in membrane biogenesis with respect to the parasitic lifestyle of Rickettsia.

Relationship between UDP-glucose 4-epimerase activity and oligoglucose glycoforms in two strains of Neisseria meningitidis.

Sodium dodecyl sulfate-polyacrylamide gel analysis of lipooligosaccharide (LOS) from Neisseria meningitidis has demonstrated considerable microheterogeneity in the variable region of LOS due to the presence of novel glycoforms. As a step toward understanding the basis for the expression of these novel glycoforms, we have examined the LOS structures and UDP-glucose 4-epimerase (epimerase) activity levels in two strains (NMB and MA-1) and their respective galE mutants. Strain NMB was found to have low epimerase activity and to contain multiple glycoforms, some of which appear to contain only glucose sugars. The galE mutant had only the oligoglucose glycoforms. Strain MA-1 had higher epimerase activity at both log and stationary phases (2- and 12.5-fold, respectively) and one glycoform with a putative lactosyl structure. Strain MA-1 galE had two glycoforms that contained one or two glucose residues. To understand the molecular basis for the different epimerase activities, we examined the predicted amino acid sequences of the respective galE open reading frames and determined the relative amounts of GalE protein. We found no significant differences between the predicted amino acid sequence of the GalE protein in NMB and that in MA-1. We observed no significant differences in the level of GalE protein between MA-1 and NMB at exponential or stationary phase. We also observed an 8.2-fold drop in epimerase activity in NMB between the log and stationary phases that was not due to the GalE protein level or low glucose levels.

Purification and some properties of UDP-N-acetylglucosamine 4-epimerase from Bacillus subtilis.

UDP-N-Acetylglucosamine 4-epimerase (EC 5.1.3.7) was purified from a cell extract of Bacillus subtilis by protamine sulfate treatment, ammonium sulfate fractionation, and column chromatographies on DEAE-Sephadex, hydroxylapatite, and Sephadex G-150. The purified enzyme was homogeneous on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 62,000 by gel filtration. The enzyme had an optimal pH in the range of 7.0 to 9.0, and was active at 30~35°C in the absence of added NAD and more active at 40~45°C in the presence of NAD. The enzyme activity was highly stimulated by small amounts of NAD. The estimated Km values were 4.4 mM for UDP-N-acetylglucosamine and 0.13mM for NAD. No UDP-glucose 4-epimerase activity was found in the purified enzyme. The enzyme was inhibited by high concentrations of glucosamine and inhibition by glucosamine was competitive with UDP-N-acetylglucosamine. The inhibition constant was 37.0 mM. N-Acetylglucosamine did not inhibit the enzyme.

Planteose Synthesis in Seeds of Sesamum indicum L.

An enzyme, UDP-galactose : sucrose 6fru-α-galactosyltransferase, which synthesizes planteose (6fru-α-galactosylsucrose), has been purified 55-fold from seeds of Sesamum indicum L. (Pedaliaceae) using ammonium sulfate and protamine sulfate precipitation. The partially purified enzyme fraction showed only very low α-galactosidase and β-fructosidase activity but significant UDP-glucose 4-epimerase, phosphodiesterase, phosphatase and sucrose synthase activity which interfere with planteose synthesis. UDP-glucose 4-epimerase activity could be inhibited by about 50% by the addition of 10 mmol/l Mn2+, whereas phosphodiesterase activity could not be reduced by the addition of ATP, NAD or UDP-glucose, potentially alternative substrates for UDP-galactose. The optimal activity of the planteose synthesizing enzyme was obtained at pH 6.2. The enzyme was stable for at least six months at -20°C. It did not need sulfhydryl reagents for full activity. The enzyme displayed a high specificity for UDP-galactose (Km 0.2–0.5 mmol/l), the galactosyl donor, and for sucrose (Km 3.6–6.0 mmol/l), the acceptor. 5′-UMP was inhibitory, while galactose 1-phosphate, glucose l-phosphate and UDP-glucose did not affect the enzyme activity.

Galactose metabolism in Dictyostelium discoideum. Regulation of galactose-1-phosphate-uridyl transferase during growth and development.

Dictyostelium discoideum is able to metabolize [1-14C]galactose to 14CO2 despite the observation that galactose is inhibitory with respect to growth. Galactose-1-phosphate uridyl transferase activity is present throughout growth and development and varies in activity only slightly during the entire life cycle of D. discoideum, in contrast to the rapid increase in UDP-glucose 4-epimerase activity during development. Therefore, in D. discoideum, these two enzymes of the Leloir pathway are independently regulated, unlike E. coli where these enzymes are coordinately controlled.