Competitive inhibition and substrate activity of uridine diphosphate 6-deoxygalactose for Escherichia coli uridine diphosphate galactose 4-epimerase.
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ABSTRACT:
SUBMITTER: Spencer M
PROVIDER: S-EPMC1177484 | biostudies-other | 1973 Feb
REPOSITORIES: biostudies-other
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Project description:1. The combined effect of the sugar nucleotides UDP-D-fucose or UDP-D-glucuronic acid together with the free sugars D-fucose or L-arabinose is the inactivation of the Escherichia coli enzyme UDP-galactose 4-epimerase (EC 5.1.3.2). The sugar nucleotide or the free sugar alone or the sugar nucleotide plus 5'-Ump do not inactivate the enzyme. 2. The inactivation of the enzyme by its substrate UDP-D-glucose was not affected by the presence of free sugar. 3. In all cases the inactivation observed follows pseudo-first-order kinetics. 4. A comparison of various sugar nucleotides indicates that the hydroxymethyl group at position 6 of the sugar moiety of the natural substrates is important for substrate binding.
| S-EPMC1172827 | biostudies-other
Project description:Kinetic and developmental characteristics of rat intestinal UDP-galactose 4-epimerase activity have been examined. The enzyme in the adult rat had a V(max.) value 2-3 times higher than that of the newborn animal, but the K(m) values for the enzyme in the newborn and adult rat were the same (0.17mm). No differences in epimerase activity were found along the length of the jejuno-ileum of adult animals, but higher activity was detected in the lower portion of the villi and crypts. The specific activity of the enzyme in the newborn rat began to rise at about 17 days of age, reaching a peak at 29 days of age, and then became constant at adult values. Total epimerase activity in the newborn rat liver was 2-5 times higher than the total activity in the intestine, and total epimerase activity in the adult intestine was 3-4 times higher than the total activity in the liver. Cortisone injection did not enhance the increase of epimerase normally seen during development, but caused a decrease in activity of this enzyme in the jejunum in rats up to 17 days of age. After 17 days, cortisone treatment had no effect on epimerase activity.
| S-EPMC1178254 | biostudies-other
Project description:Trypanosoma brucei, the causative agent of human African trypanosomiasis, affects tens of thousands of sub-Saharan Africans. As current therapeutics are inadequate due to toxic side effects, drug resistance, and limited effectiveness, novel therapies are urgently needed. UDP-galactose 4'-epimerase (TbGalE), an enzyme of the Leloir pathway of galactose metabolism, is one promising T. brucei drug target. We here use the relaxed complex scheme, an advanced computer-docking methodology that accounts for full protein flexibility, to identify inhibitors of TbGalE. An initial hit rate of 62% was obtained at 100 microM, ultimately leading to the identification of 14 low-micromolar inhibitors. Thirteen of these inhibitors belong to a distinct series with a conserved binding motif that may prove useful in future drug design and optimization.
| S-EPMC2895357 | biostudies-literature
Project description:High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48 mM (27.2 g/L) in a small-scale batch process (200 μL) within 24 h using 0.02 genzyme /gproduct . Through in-situ ATP regeneration, the amount of ATP (0.6 mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1 L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4 g/L UDP-galactose were produced within 23 h with a synthesis yield of 71 % and a biocatalyst load of 0.05 gtotal_protein /gproduct . The costs for substrates per gram of UDP-galactose synthesized were around 0.26 €/g.
| S-EPMC9299652 | biostudies-literature
Project description:The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD(+) and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the K m and k cat for the enzyme were determined to be 0.11 mM and 12.8 s(-1), respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results.
| S-EPMC3792076 | biostudies-literature
Project description:The following observations showed that 2-deoxy-D-galactose is a useful tool for the isolation and elucidation of the activity of one system for galactose uptake into Escherichia coli. 1. 2-Deoxygalactose, which is not a substrate for growth of E. coli, was transported into strains of the organism induced for galactose transport. 2. By using appropriate mutants it was shown that 2-deoxygalactose is a much better substrate for the galactose-transport system than for the methyl galactoside-transport system. This was confirmed by the results of mutual inhibition studies with substrates of each transport system. 3. The glucose-, arabinose- or lactose-transport systems did not effect significant transport of 2-deoxygalactose. 4. Like other substrates of the galactose-transport system, 2-deoxygalactose promoted effective proton uptake into de-energized suspensions of appropriate E. coli strains. 5. The S183 series of E. coli mutants were found to contain a constitutive galactose-transport system, if 2-deoxygalactose transport is used as one criterion for such activity.
| S-EPMC1184112 | biostudies-other
Project description:1. The enzyme that catalyses the transfer of galactose from UDP-galactose to N-acetylgalactosaminyl-(1-->4)-N-acetylneuraminyl-(2-->3)-galactosyl-(1-->4)-glucosylceramide (G(M2)) was found mainly in the heavy- and light-microsomal fractions of the adult frog brain. 2. The subcellular distribution of the enzyme, UDP-galactose-G(M2) galactosyltransferase, parallels that of gangliosides in adult frog brain. 3. The enzymic activity was first detected at late gastrulation (Shumway stage 11(1/2)) and increased until the completion of the operculum (Shumway stage 25) and then decreased in the tadpoles. 4. In adult frog brain, the enzyme exhibited a pH optimum of 7.2-7.3 in both cacodylate and tris buffers. The enzyme required 10mm-Mn(2+) for maximal activity and the K(m) for Mn(2+) was determined as 2.2mm. The half-maximal velocity was obtained at a G(M2) concentration of 0.18mm. Inhibition of the enzymic reaction was found when the G(M2) concentration was greater than 1.38mm. 5. The enzymic activity was also inhibited by the products in the pathway of ganglioside synthesis, i.e. either by a mixture of gangliosides or by individual ganglioside components. The most active inhibitor was disialoganglioside. The degree of inhibition is a function of the individual ganglioside concentration. 6. A product-inhibition mechanism for the regulation of ganglioside biosynthesis is discussed.
| S-EPMC1179110 | biostudies-other
Project description:BackgroundGinsenosides are known as the principal pharmacological active constituents in Panax medicinal plants such as Asian ginseng, American ginseng, and Notoginseng. Some ginsenosides, especially the 20(R) isomers, are found in trace amounts in natural sources and are difficult to chemically synthesize. The present study provides an approach to produce such trace ginsenosides applying biotransformation through Escherichia coli modified with relevant genes.MethodsSeven uridine diphosphate glycosyltransferase (UGT) genes originating from Panax notoginseng, Medicago sativa, and Bacillus subtilis were synthesized or cloned and constructed into pETM6, an ePathBrick vector, which were then introduced into E. coli BL21star (DE3) separately. 20(R)-Protopanaxadiol (PPD), 20(R)-protopanaxatriol (PPT), and 20(R)-type ginsenosides were used as substrates for biotransformation with recombinant E. coli modified with those UGT genes.ResultsE. coli engineered with GT95 syn selectively transfers a glucose moiety to the C20 hydroxyl of 20(R)-PPD and 20(R)-PPT to produce 20(R)-CK and 20(R)-F1, respectively. GTK1- and GTC1-modified E. coli glycosylated the C3-OH of 20(R)-PPD to form 20(R)-Rh2. Moreover, E. coli containing p2GT95synK1, a recreated two-step glycosylation pathway via the ePathBrich, implemented the successive glycosylation at C20-OH and C3-OH of 20(R)-PPD and yielded 20(R)-F2 in the biotransformation broth.ConclusionThis study demonstrates that rare 20(R)-ginsenosides can be produced through E. coli engineered with UTG genes.
| S-EPMC6323243 | biostudies-literature
Project description:1. Lactose synthetase activity in the rat mammary gland increases during the last day of pregnancy from an essentially zero value. There is a parallel increase of tissue lactose and of glucose 6-phosphate dehydrogenase activity. 2. Mammary-gland homogenates prepared both before and after parturition hydrolyse the lactose precursors glucose 6-phosphate, glucose 1-phosphate, UDP-glucose, UDP-galactose and also maltose, but not lactose. 3. A role of lactose synthetase as the rate-limiting enzyme for lactose biosynthesis and the possible significance of the hydrolytic activities are discussed with respect to lactogenesis.
| S-EPMC1198567 | biostudies-other
Project description:Galactose epimerase deficiency is an inborn error of metabolism due to uridine diphosphate-galactose-4'-epimerase (GALE) deficiency. We report the clinical presentation, genetic and biochemical studies in two siblings with generalized GALE deficiency.Patient 1: The first child was born with a dysmorphic syndrome. Failure to thrive was noticed during the first year. Episodes of heart failure due to dilated cardiomyopathy, followed by liver failure, occurred between 12 and 42 months. The finding of a serum transferrin isoelectrofocusing (IEF) type 1 pattern led to the suspicion of a congenital disorder of glycosylation (CDG). Follow-up disclosed psychomotor disability, deafness, and nuclear cataracts.Patient 2: The sibling of patient 1 was born with short limbs and hip dysplasia. She is deceased in the neonatal period due to intraventricular hemorrhage in the context of liver failure. Investigation disclosed galactosuria and normal transferrin glycosylation.Next-generation sequence panel analysis for CDG syndrome revealed the previously reported c.280G>A (p.[V94M]) homozygous mutation in the GALE gene. Enzymatic studies in erythrocytes (patient 1) and fibroblasts (patients 1 and 2) revealed markedly reduced GALE activity confirming generalized GALE deficiency. This report describes the fourth family with generalized GALE deficiency, expanding the clinical spectrum of this disorder, since major cardiac involvement has not been reported before.
| S-EPMC5740041 | biostudies-literature