Cloning and expression analysis of a UDP-galactose/glucose pyrophosphorylase from melon fruit provides evidence for the major metabolic pathway of galactose metabolism in raffinose oligosaccharide metabolizing plants

The Cucurbitaceae translocate a significant portion of their photosynthate as raffinose and stachyose, which are galactosyl derivatives of sucrose. These are initially hydrolyzed by alpha-galactosidase to yield free galactose (Gal) and, accordingly, Gal metabolism is an important pathway in Cucurbitaceae sink tissue. We report here on a novel plant-specific enzyme responsible for the nucleotide activation of phosphorylated Gal and the subsequent entry of Gal into sink metabolism. The enzyme was antibody purified, sequenced, and the gene cloned and functionally expressed in Escherichia coli. The heterologous protein showed the characteristics of a dual substrate UDP-hexose pyrophosphorylase (PPase) with activity toward both Gal-1-P and glucose (Glc)-1-P in the uridinylation direction and their respective UDP-sugars in the reverse direction. The two other enzymes involved in Glc-P and Gal-P uridinylation are UDP-Glc PPase and uridyltransferase, and these were also cloned, heterologously expressed, and characterized. The gene expression and enzyme activities of all three enzymes in melon (Cucumis melo) fruit were measured. The UDP-Glc PPase was expressed in melon fruit to a similar extent as the novel enzyme, but the expressed protein was specific for Glc-1-P in the UDP-Glc synthesis direction and did not catalyze the nucleotide activation of Gal-1-P. The uridyltransferase gene was only weakly expressed in melon fruit, and activity was not observed in crude extracts. The results indicate that this novel enzyme carries out both the synthesis of UDP-Gal from Gal-1-P as well as the subsequent synthesis of Glc-1-P from the epimerase product, UDP-Glc, and thus plays a key role in melon fruit sink metabolism.     

Leishmania UDP-sugar Pyrophosphorylase: THE MISSING LINK IN GALACTOSE SALVAGE?

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.     

Carbohydrate metabolism during early fruit development of sweet melon (Cucumis melo)

In sink tissues of cucurbits, including sweet melon fruits, the galactosyl-sucrose oligosaccharides, stachyose and raffinose, together with sucrose, are the major translocated carbohydrates. In the present study we investigated the carbohydrate metabolism of young melon ( Cucumis melo L. cv. C-8) fruit during the period of initial fruit set and development, from 3 days prior to anthesis until 20 days after anthesis (DAA), prior to the onset of sucrose accumulation. The enzymes assayed could be classified into two categories according to developmental patterns. Two of the enzymes, alkaline α -galactosidase I [EC 3.2.1.22], which hydrolyzes both raffinose and stachyose, and acid invertase [EC 3.2.1.26] either increased or remained stable during the first 10 DAA. The remaining measured enzymes (the stachyose-specific alkaline α -galactosidase form II, acid α -galactosidase, alkaline invertase, sucrose synthase [EC 2.4.1.13], galactokinase [EC 2.7.1.6], UDP-Gal PPase [EC 2.7.7.10], UDP-Glc-4 epimerase [EC 5.1.3.2], UDP-Glc PPase [EC 2.7.7.9], phosphoglucomutase [EC 5.4.2.2] and phosphoglucoisomerase [EC 5.3.1.9]) all showed a similar developmental pattern of steady decrease in activity following anthesis. We also compared the saccharide metabolism of pollinated and non-pollinated ovaries during the initial days following anthesis. In the absence of pollination, ovary growth dramatically decreased by the first DAA and was accompanied by a sharp decrease in the activity of UDP-Glc PPase. Other enzymes in the pathway, including the enzymes of stachyose and raffinose hydrolysis, did not decrease in activity until 2 or 4 DAA, after ovary growth was affected. These results provide information to assess the possible regulating enzymes in cucurbit ovary development and fruit set.     

Purification and characterization of the raffinose oligosaccharide chain elongation enzyme,galactan : galactan galactosyltransferase (GGT), from Ajuga reptans leaves

Galactan: galactan galactosyltransferase (GGT), an enzyme involved in the biosynthesis of the long-chain raffinose family of oligosaccharides (RFOs) in Ajuga reptans, catalyses the transfer of an alpha-galactosyl residue from one molecule of RFO to another one resulting in the next higher RFO oligomer. This novel galactinol (alpha-galactosyl-myo-inositol)-independent alpha-galactosyltransferase is responsible for the accumulation of long-chain RFOs in vivo. Warm treatment (20 degrees C) of excised leaves resulted in a 34-fold increase of RFO concentration and a 200-fold increase of GGT activity after 28 days. Cold treatment (10 degrees C/3 degrees C day/night) resulted in a 26- and 130-fold increase, respectively. These data support the role of GGT as a key enzyme in the synthesis and accumulation of long-chain RFOs. GGT was purified from leaves in a 4-step procedure which involved fractionated precipitation with ammonium sulphate as well as lectin affinity, anion exchange, and size-exclusion chromatography and resulted in a 200-fold purification. Purified GGT had an isoelectric point of 4.7, a pH optimum around 5, and its transferase reaction displayed saturable concentration dependence for both raffinose (Km = 42 mM) and stachyose (Km = 58 mM). GGT is a glycoprotein with a 10% glycan portion. The native molecular mass was 212 kDa as determined by size-exclusion chromatography. Purified GGT showed one single active band after native PAGE or IEF separation, respectively, which separated into three bands on SDS-PAGE at 48 kDa, 66 kDa, and 60 kDa. The amino acid sequence of four tryptic peptides obtained from the major 48-kDa band showed a high homology to plant alpha-galactosidase (EC 3.2.1.22) sequences. GGT differed, however, in its substrate specificity from alpha-galactosidases; it neither hydrolysed nor transferred alpha-galactosyl-groups from melibiose, galactinol, UDP-galactose, manninotriose, and manninotetrose. Galactinol, sucrose, and galactose inhibited the GGT reaction considerably at 10-50 mM.     

Galactinol synthase activity and soluble sugars in developing seeds of four soybean genotypes

Galactinol synthase (UDP-galactose:inositol galactosyltransferase) is the first unique enzyme in the biosynthetic pathway of raffinose saccharides. Its role as a regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean (Glycine max L. Merrill) seeds was examined. Galactinol synthase activity and concentrations of sucrose, stachyose, and raffinose were compared during seed development between two genotypes that were high and two genotypes that were low in mature seed raffinose saccharide concentration. In all genotypes, sucrose concentration increased as seed development progressed, but in both low raffinose saccharide genotypes, greater increases in sucrose concentration were observed late in seed development. Sucrose to stachyose ratios in mature seeds were 2.3-fold greater in low raffinose saccharide genotypes than in the high raffinose saccharide genotypes. During seed development, higher levels of galactinol synthase activity were observed in the high raffinose saccharide genotypes than in the low raffinose saccharide genotypes. A common linear relationship for all four soybean genotypes was shown to exist between galactinol formed estimated from galactinol synthase activity data and the concentration of galactose present in raffinose saccharides. Results of this study implied that galactinol synthase is an important regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean seeds.     

Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-glucose and UDP-galactose during normal galactose metabolism. In humans, deficiencies in this enzyme lead to the complex disorder referred to as epimerase-deficiency galactosemia. Here, we describe the high-resolution X-ray crystallographic structures of human epimerase in the resting state (i.e., with bound NAD(+)) and in a ternary complex with bound NADH and UDP-glucose. Those amino acid side chains responsible for anchoring the NAD(+) to the protein include Asp 33, Asn 37, Asp 66, Tyr 157, and Lys 161. The glucosyl group of the substrate is bound to the protein via the side-chain carboxamide groups of Asn 187 and Asn 207. Additionally, O(gamma) of Ser 132 and O(eta) of Tyr 157 lie within 2.4 and 3.1 A, respectively, of the 4'-hydroxyl group of the sugar. Comparison of the polypeptide chains for the resting enzyme and for the protein with bound NADH and UDP-glucose demonstrates that the major conformational changes which occur upon substrate binding are limited primarily to the regions defined by Glu 199 to Asp 240 and Gly 274 to Tyr 308. Additionally, this investigation reveals for the first time that a conserved tyrosine, namely Tyr 157, is in the proper position to interact directly with the 4'-hydroxyl group of the sugar substrate and to thus serve as the active-site base. A low barrier hydrogen bond between the 4'-hydroxyl group of the sugar and O(gamma) of Ser 132 facilitates proton transfer from the sugar 4'-hydroxyl group to O(eta) of Tyr 157.     

The key enzyme in galactose metabolism, UDP-galactose-4-epimerase, affects cell-wall integrity and morphology in Candida albicans even in the absence of galactose

The enzyme UDP-galactose-4-epimerase (GAL10) catalyzes a key step in galactose metabolism converting UDP-galactose to UDP-glucose which then can get metabolized through glycolysis and TCA cycle thus allowing the cell to use galactose as a carbon and energy source. As in many fungi, a functional homolog of GAL10 exists in Candida albicans. The domainal organization of the homologs from Saccharomyces cerevisiae and C. albicans show high degree of homology having both mutarotase and an epimerase domain. The former is responsible for the conversion of beta-d-galactose to alpha-d-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. Absence of C. albicans GAL10 (CaGAL10) affects cell-wall organization, oxidative stress response, biofilm formation and filamentation. Cagal10 mutant cells tend to flocculate extensively as compared to the wild-type cells. The excessive filamentation in this mutant is reflected in its irregular and wrinkled colony morphology. Cagal10 strain is more susceptible to oxidative stress when tested in presence of H2O2. While the S. cerevisiae GAL10 (ScGAL10), essential for survival in the presence of galactose, has not been reported to have defects in the absence of galactose, the C. albicans homolog shows these phenotypes during growth in the absence of galactose. Thus a functional CaGal10 is required not only for galactose metabolism but also for normal hyphal morphogenesis, colony morphology, maintenance of cell-wall integrity and for resistance to oxidative stress even in the absence of galactose.     

Purification and properties of UDP-glucose (UDP-galactose) pyrophosphorylase from Bifidobacterium bifidum.

An enzyme having both UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) pyrophosphorylase activities was purified to homogeneity from Bifidobacterium bifidum. The molecular weight of the enzyme was about 200,000 and it appeared to be composed of four identical subunits. The purified enzyme showed almost the same reactivity towards UDP-Glc and UDP-Gal, and showed about 10% of this activity towards UDP-xylose at 8 mM. The enzyme required magnesium ions for maximum activity. The apparent equilibrium constants were about 2.5 for UDP-Glc pyrophosphorolysis and 1.1 for UDP-Gal pyrophosphorolysis. The enzyme activities were inhibited by various nucleotides (product or substrate analogs). Some sugar phosphates, such as fructose 6-P, erythrose 4-P, and 3-phosphoglycerate, stimulated the activities. These properties are discussed in relation to the significance of the enzyme in galactose metabolism of B. bifidum.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: analysis of its hysteretic behavior during catalysis

UDP-galactose 4-epimerase serves as a prototype model of class II oxidoreductases that use bound NAD as a cofactor. This enzyme from Kluyveromyces fragilis is a homodimer with a molecular mass of 75 kDa/subunit. Continuous monitoring of the conversion of UDP-galactose (UDP-gal) to UDP-glucose (UDP-glu) by the epimerase in the presence of the coupling enzyme UDP-glucose dehydrogenase and NAD shows a kinetic lag of up to 80 s before a steady state is reached. The disappearance of the lag follows first-order kinetics (k = 3.22 x 10(-2) s(-1)) at 25 degrees C at enzyme and substrate concentrations of 1.0 nM and 1 mM, respectively. The observed lag is not due to factors such as insufficient activity of the coupling enzyme, association or dissociation or incomplete recruitment of NAD by epimerase, product activation, etc., but was a true expression of the activity of the prepared enzyme. Dissociation of the bound ligand(s) by heat followed by analysis with reverse-phase HPLC, TLC, UV-absorption spectrometry, mass spectrometry, and NMR showed that in addition to 1.78 mol of NAD/dimer, the epimerase also contains 0.77 mol of 5'-UMP/dimer. The latter is a strong competitive inhibitor. Preincubation of the epimerase with the substrate UDP-gal or UDP-glu replaces the inhibitor and also abolishes the lag, which reappeared after the enzyme was treated with 5'-UMP. The lag was not observed as long as the cells were in the growing phase and galactose in the growth medium was limiting, suggesting that association with 5'-UMP is a late log-phase phenomenon. The stoichiometry and conserved amino acid sequence around the NAD binding site of multimeric class I (classical dehydrogenases) and class II oxidoreductases, as reported in the literature, have been compared. It shows that each subunit is independently capable of being associated with one molecule of NAD, suggestive of two NAD binding sites of epimerase per dimer.     

Sucrose Metabolism in Netted Muskmelon Fruit during Development

Sugar content and composition are major criteria used in judging the quality of netted muskmelon (Cucumis melo L. var reticulatus) fruit. Sugar composition and four enzymes of sucrose metabolism were determined in `Magnum 45' muskmelon fruit at 10-day intervals beginning 10 days after pollination (DAP) until full-slip (35 DAP). Sugar content increased in both outer (green) mesocarp and inner (orange) mesocarp between 20 and 30 DAP. The major proportion of total increase in sugar was attributed to sucrose accumulation. The large increase in sucrose relative to glucose and fructose was accompanied by a dramatic decrease in acid invertase activity, which was highest in both tissues at 10 and 20 DAP, and increases in sucrose phosphate synthase (SPS) and sucrose synthase activities. The green tissue had a lower proportion of total sugar as sucrose, greater invertase activity, and less SPS activity than the orange tissue. Changes in relative sucrose content were highly correlated with changes in enzyme activity. The results strongly suggest that increases in the proportion of sucrose found in melon fruit were associated with a decline in acid invertase activity and an increase in SPS activity approximately 10 days before full-slip. Therefore, these enzymes apparently play a key role in determining sugar composition and the quality of muskmelon fruit.     

Epimerase-deficiency galactosemia is not a binary condition

Epimerase-deficiency galactosemia results from the impairment of UDP-galactose 4'-epimerase (GALE), the third enzyme in the Leloir pathway of galactose metabolism. Originally identified as a clinically benign "peripheral" condition with enzyme impairment restricted to circulating blood cells, GALE deficiency was later demonstrated also to exist in a rare but clinically severe "generalized" form, with enzyme impairment affecting a range of tissues. Isolated cases of clinically and/or biochemically intermediate cases of epimerase deficiency have also been reported. We report here studies of 10 patients who, in the neonatal period, received the diagnosis of hemolysate epimerase deficiency. We have characterized these patients with regard to three parameters: (1) GALE activity in transformed lymphoblasts, representing a "nonperipheral" tissue, (2) metabolic sensitivity of those lymphoblasts to galactose challenge in culture, and (3) evidence of normal versus abnormal galactose metabolism in the patients themselves. Our results demonstrate two important points. First, whereas some of the patients studied exhibited near-normal levels of GALE activity in lymphoblasts, consistent with a diagnosis of peripheral epimerase deficiency, many did not. We detected a spectrum of GALE activity levels ranging from 15%-64% of control levels, demonstrating that epimerase deficiency is not a binary condition; it is a continuum disorder. Second, lymphoblasts demonstrating the most severe reduction in GALE activity also demonstrated abnormal metabolite levels in the presence of external galactose and, in some cases, also in the absence of galactose. These abnormalities included elevated galactose-1P, elevated UDP-galactose, and deficient UDP-glucose. Moreover, some of the patients themselves also demonstrated metabolic abnormalities, both on and off galactose-restricted diet. Long-term follow-up studies of these and other patients will be required to elucidate the clinical significance of these biochemical abnormalities and the potential impact of dietary intervention on outcome.     

Transport of UDP-galactose in plants. Identification and functional characterization of AtUTr1, an Arabidopsis thaliana UDP-galactos/UDP-glucose transporter

The synthesis of non-cellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as substrate. The topology of these reactions is not known, although the orientation of a plant galactosyltransferase involved in the biosynthesis of galactomannans in fenugreek is consistent with a requirement for UDP-galactose in the lumen of the Golgi cisternae. Here we provide evidence that sealed, right-side-out Golgi vesicles isolated from pea stems transport UDP-galactose into their lumen and transfer galactose, likely to polysaccharides and other acceptors. In addition, we identified and cloned AtUTr1, a gene from Arabidopsis thaliana that encodes a multitransmembrane hydrophobic protein similar to nucleotide sugar transporters. Northern analysis showed that AtUTr1 is indeed expressed in Arabidopsis. AtUTr1 is able to complement the phenotype of MDCK ricin-resistant cells; a mammalian cell line deficient in transport of UDP-galactose into the Golgi. In vitro assays using a Golgi-enriched vesicle fraction obtained from Saccharomyces cerevisiae expressing AtUTr1-MycHis is able to transport UDP-galactose but also UDP-glucose. AtUTr1- MycHis does not transport GDP-mannose, GDP-fucose, CMP-sialic acid, UDP-glucuronic acid, or UDP-xylose when expressed in S. cerevisiae. AtUTr1 is the first transporter described that is able to transport UDP-galactose and UDP-glucose. Thus AtUTr1 may play an important role in the synthesis of glycoconjugates in Arabidopsis that contain galactose and glucose.     

Molecular cloning of the Leishmania major UDP-glucose pyrophosphorylase, functional characterization, and ligand binding analyses using NMR spectroscopy

The dense glycocalyx surrounding the protozoan parasite Leishmania is an essential virulence factor. It protects the parasite from hostile environments in the sandfly vector and mammalian host and supports steps of development and invasion. Therefore, new therapeutic concepts concentrate on disturbing glycocalyx biosynthesis. Deletion of genes involved in the metabolism of galactose and mannose have been shown to drastically reduce Leishmania virulence. Here we report the identification of Leishmania major UDP-glucose pyrophosphorylase (UGP). UGP catalyzes the formation of UDP-glucose from glucose 1-phosphate and UTP. This activation step enables glucose to enter metabolic pathways and is crucial for the activation of galactose. UDP-galactose is made from UDP-glucose by nucleotide-donor transfer to galactose 1-phosphate or by epimerization of the glucose moiety. Isolated in a complementation cloning approach, the activity of L. major UGP was proven in vitro. Moreover, purified protein was used to investigate enzyme kinetics, quaternary organization, and binding of ligands. Whereas sequestration by oligomerization is a known regulatory mechanism for eukaryotic UGPs, the recombinant as well as native L. major UGP migrated as monomer in size exclusion chromatography and in accord with this showed simple Michaelis-Menten kinetics toward all substrates. In saturation transfer difference (STD)-NMR studies, we clearly demonstrated that the molecular geometry at position 4 of glucose is responsible for substrate specificity. Furthermore, the gamma-phosphate group of UTP is essential for binding and for induction of the open conformation, which then allows entry of glucose 1-phosphate. Our data provide the first direct proof for the ordered bi-bi mechanism suggested in earlier studies.     

Mediators of galactose sensitivity in UDP-galactose 4'-epimerase-impaired mammalian cells

UDP-galactose 4'-epimerase (GALE) catalyzes the final step in the Leloir pathway of galactose metabolism, interconverting UDP-galactose and UDP-glucose. Unlike its Escherichia coli counterpart, mammalian GALE also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. Considering the key roles played by all four of these UDP-sugars in glycosylation, human GALE therefore not only contributes to the Leloir pathway, but also functions as a gatekeeper overseeing the ratios of important substrate pools required for the synthesis of glycosylated macromolecules. Defects in human GALE result in the disorder epimerase-deficiency galactosemia. To explore the relationship among GALE activity, substrate specificity, metabolic balance, and galactose sensitivity in mammalian cells, we employed a previously described GALE-null line of Chinese hamster ovary cells, ldlD. Using a transfection protocol, we generated ldlD derivative cell lines that expressed different levels of wild-type human GALE or E. coli GALE and compared the phenotypes and metabolic profiles of these lines cultured in the presence versus absence of galactose. We found that GALE-null cells accumulated abnormally high levels of Gal-1-P and UDP-Gal and abnormally low levels of UDP-Glc and UDP-GlcNAc in the presence of galactose and that human GALE expression corrected each of these defects. Comparing the human GALE- and E. coli GALE-expressing cells, we found that although GALE activity toward both substrates was required to restore metabolic balance, UDP-GalNAc activity was not required for cell proliferation in the presence of otherwise cytostatic concentrations of galactose. Finally, we found that uridine supplementation, which essentially corrected UDP-Glc and, to a lesser extent UDP-GlcNAc depletion, enabled ldlD cells to proliferate in the presence of galactose despite the continued accumulation of Gal-1-P and UDP-Gal. These data offer important insights into the mechanism of galactose sensitivity in epimerase-impaired cells and suggest a potential novel therapy for patients with epimerase-deficiency galactosemia.     

High resolution x-ray structure of tyvelose epimerase from Salmonella typhi

Tyvelose epimerase catalyzes the last step in the biosynthesis of tyvelose by converting CDP-d-paratose to CDP-d-tyvelose. This unusual 3,6-dideoxyhexose occurs in the O-antigens of some types of Gram-negative bacteria. Here we describe the cloning, protein purification, and high-resolution x-ray crystallographic analysis of tyvelose epimerase from Salmonella typhi complexed with CDP. The enzyme from S. typhi is a homotetramer with each subunit containing 339 amino acid residues and a tightly bound NAD+ cofactor. The quaternary structure of the enzyme displays 222 symmetry and can be aptly described as a dimer of dimers. Each subunit folds into two distinct lobes: the N-terminal motif responsible for NAD+ binding and the C-terminal region that harbors the binding site for CDP. The analysis described here demonstrates that tyvelose epimerase belongs to the short-chain dehydrogenase/reductase superfamily of enzymes. Indeed, its active site is reminiscent to that observed for UDP-galactose 4-epimerase, an enzyme that plays a key role in galactose metabolism. Unlike UDP-galactose 4-epimerase where the conversion of configuration occurs about C-4 of the UDP-glucose or UDP-galactose substrates, in the reaction catalyzed by tyvelose epimerase, the inversion of stereochemistry occurs at C-2. On the basis of the observed binding mode for CDP, it is possible to predict the manner in which the substrate, CDP-paratose, and the product, CDP-tyvelose, might be accommodated within the active site of tyvelose epimerase.     

Galactose starvation in a bloodstream form Trypanosoma brucei UDP-glucose 4'-epimerase conditional null mutant

Galactose metabolism is essential for the survival of Trypanosoma brucei, the etiological agent of African sleeping sickness. T. brucei hexose transporters are unable to transport galactose, which is instead obtained through the epimerization of UDP-glucose to UDP-galactose catalyzed by UDP-glucose 4'-epimerase (galE). Here, we have characterized the phenotype of a bloodstream form T. brucei galE conditional null mutant under nonpermissive conditions that induced galactose starvation. Cellular levels of UDP-galactose dropped rapidly upon induction of galactose starvation, reaching undetectable levels after 72 h. Analysis of extracted glycoproteins by ricin and tomato lectin blotting showed that terminal beta-d-galactose was virtually eliminated and poly-N-acetyllactosamine structures were substantially reduced. Mass spectrometric analysis of variant surface glycoprotein confirmed complete loss of galactose from the glycosylphosphatidylinositol anchor. After 96 h, cell division ceased, and electron microscopy revealed that the cells had adopted a morphologically distinct stumpy-like form, concurrent with the appearance of aberrant vesicles close to the flagellar pocket. These data demonstrate that the UDP-glucose 4'-epimerase is essential for the production of UDP-galactose required for galactosylation of glycoproteins and that galactosylation of one or more glycoproteins, most likely in the lysosomal/endosomal system, is essential for the survival of bloodstream form T. brucei.     

Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose during normal galactose metabolism. One of the key structural features in the proposed reaction mechanism for the enzyme is the rotation of a 4'-ketopyranose intermediate within the active site pocket. Recently, the three-dimensional structure of the human enzyme with bound NADH and UDP-glucose was determined. Unlike that observed for the protein isolated from Escherichia coli, the human enzyme can also turn over UDP-GlcNAc to UDP-GalNAc and vice versa. Here we describe the three-dimensional structure of human epimerase complexed with NADH and UDP-GlcNAc. To accommodate the additional N-acetyl group at the C2 position of the sugar, the side chain of Asn-207 rotates toward the interior of the protein and interacts with Glu-199. Strikingly, in the human enzyme, the structural equivalent of Tyr-299 in the E. coli protein is replaced with a cysteine residue (Cys-307) and the active site volume for the human protein is calculated to be approximately 15% larger than that observed for the bacterial epimerase. This combination of a larger active site cavity and amino acid residue replacement most likely accounts for the inability of the E. coli enzyme to interconvert UDP-GlcNAc and UDP-GalNAc.     

The biosynthesis of raffinose

1. Reaction of UTP and alpha-d-galactose 1-phosphate with [U-(14)C]sucrose in the presence of a Vicia faba dormant-seed preparation yielded the trisaccharide raffinose. 2. UTP-alpha-d-galactose 1-phosphate-uridylyltransferase activity has been demonstrated in the bean preparation and evidence for the participation of UDP-galactose in the trisaccharide synthesis is presented. 3. UDP-galactose 4-epimerase is present in the dormant seed. 4. The biosynthesis of raffinose in relation to the metabolism of other carbohydrates in plants is discussed.     

Studies on the metabolic determinants of D-galactose-induced neurotoxicity in the chick

The contents of galactose, galactitol, galactose 1-phosphate, UDP-galactose and UDP-glucose in the brains of chicks fed a diet containing 40 % (w/w) D-galactose were determined at regular intervals during a 48 h period which terminated in convulsive activity and death of the animals. Although levels of galactose and galactitol were markedly elevated, UDP-galactose and UDP-glucose levels were not significantly increased. The level of galactose 1-phosphate rose to 1-3 μg/g of fresh tissue by 14 h but gradually diminished until, at 48 h, the content was 0-25 μg/g. The metabolic turnover of these compounds, as shown by labelling experiments with inorganic [³²P]phosphate and [U-¹⁴C]galactose, indicated that galactose 1-phosphate and UDP-galactose were rapidly metabolized, yet relatively little galactose was utilized by the brain as a source of energy. These observations have prompted us to propose a mechanism for the turnover of galactose 1-phosphate that involves cyclical phosphorylation and dephosphorylation reactions in the brains of galactose-fed chicks. In support of this hypothesis, we have identified phosphatase activity which has a relatively low K_m value for galactose 1-phosphate (0-06-0-07 mM) in virtually all subcellular fractions of homogenates of chick brain. Maximum activity of the phosphatase is several-fold greater than that recorded for galactokinase (EC 2.7.1.6) and galactose 1-phosphate uridyltransferase (EC 2.7.710) from chicken brain.     

Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone

We used homologous and heterologous expression of the glycosyltransferase genes of the Lactococcus lactis NIZO B40 eps gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.