Synthesis Of UDP-galactose Research Articles

One-step synthesis of sugar nucleotides under optimized conditions

Sugar nucleotides are essential substrates used for synthesizing bioactive oligosaccharides in enzymatic reactions, however, their preparation via chemical and enzyme reactions is challenging. Therefore, we investigated the synthesis of sugar nucleotides under optimal conditions using a facile one-step reaction. Seven sugar nucleotides were synthesized via an optimized one-step reaction in 40–80% yield, exhibiting a preference for 1,2-trans glycoside. Moreover, a gram-scale synthesis of UDP-galactose resulted in a 46% (578 mg) yield.

Heterologous expression of a novel galactose-1-phosphate uridylyltransferase from Thermodesulfatator indicus and its application for bioproduction of Gal-β-1,4-GlcNAc-X

Nucleotide sugars (UDP-Sugars) are essential for the production of polysaccharides and glycoconjugates utilized in medicines, cosmetics, and food industries. The enzyme Galactose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.12) is responsible for the synthesis of UDP-galactose from α-d-galactose-1-phosphate (Gal-1P) and UTP. A novel bacterial GalU (TiGalU) encoded from a thermophilic bacterium, Thermodesulfatator indicus, was successfully purified using the Ni-NTA column after being expressed in Escherichia coli. The optimal pH for recombinant TiGalU was determined to be 5.5. The optimum temperature of the enzyme was 45 °C. The activity of TiGalU was not dependent on Mg2+ and was strongly inhibited by SDS. When coupled with galactose kinase (GALK1) and β-1,4-galactosyltransferase 1 (B4GALT1), the enzyme enabled the one-pot synthesis of Gal-β-1,4-GlcNAc-X by utilizing galactose and UTP as substrates. This study reported the in vitro biosynthesis of Gal-β-1,4-GlcNAc-X for the first time, providing an environmentally friendly way to biosynthesis glycosides and other polysaccharides.

High-Level Production of Lacto-N-neotetraose in Escherichia coli by Stepwise Optimization of the Biosynthetic Pathway.

Lacto-N-neotetraose (LNnT), an abundant human milk oligosaccharide (HMO), has been approved as a novel functional additive for infant formulas. Therefore, LNnT biosynthesis has attracted extensive attention. Here, a high LNnT-producing, low lacto-N-triose II (LNT II)-residue Escherichia coli strain was constructed. First, an initial LNnT-producing chassis strain was constructed by blocking lactose, UDP-N-acetylglucosamine, and UDP-galactose competitive consumption pathways and introducing β-1,3-N-acetylglucosaminyltransferase LgtA and β-1,4-galactosyltransferase LgtB. Subsequently, the supply of LNnT precursors was increased by enhancing UDP-N-acetylglucosamine and UDP-galactose synthesis, inactivating LNT II extracellular transporter SetA, and improving UTP synthesis. Then, modular engineering strategy was used to optimize LNnT biosynthetic pathway fluxes. Moreover, pathway fluxes were fine-tuned by modulating translation initiation strength of essential genes lgtB, prs, and lacY. Finally, LNnT production reached 6.70 g/L in a shake flask and 19.40 g/L in a 3 L bioreactor with 0.47 g/(L h) productivity, with 1.79 g/L LNT II residue, highest productivity level, and lowest LNT II residue thus far.

Role of AlgC and GalU in the Intrinsic Antibiotic Resistance of Helicobacter pylori.

Helicobacter pylori is associated with the development of gastrointestinal diseases. However, its eradication is challenged by an increased rate of drug resistance. AlgC and GalU are important for the synthesis of UDP-glucose, which is a substrate for the synthesis of lipopolysaccharide (LPS) in H. pylori. In this study, we investigated the role of UDP-glucose in the intrinsic drug resistance in H. pylori. Gene knockout strains or complementation strains, including ΔalgC, ΔgalU, ΔgalE, Δhp0045, ΔalgC/algC* and ΔgalU/galU* were constructed in Hp26695; and ΔalgC and ΔgalU were also constructed in two clinical drug-resistant strains, Hp008 and Hp135. The minimum inhibitory concentrations (MIC) of H. pylori to amoxicillin (AMO), tetracycline (TET), clarithromycin (CLA), metronidazole (MNZ), levofloxacin (LEV), and rifampicin (RIF) were measured using MIC Test Strips. Silver staining was performed to examine the role of AlgC and GalU in LPS synthesis. Ethidium bromide (EB) accumulation assay was performed to assess the outer membrane permeability of H. pylori strains. Knockout of algC and galU in H. pylori resulted in increased drug sensitivity to AMO, MNZ, CLA, LEV, and RIF; whereas knockout of hp0045 and galE, which are involved in GDP-fucose and UDP-galactose synthesis, respectively, did not significantly alter the drug sensitivity of H. pylori. Knockout of algC and galU in clinically drug-resistant strains resulted in significantly increased drug sensitivity to all the antibiotics, except MNZ. The lipid A-core structure was altered in ΔalgC and ΔgalU when their EB accumulation was higher than that in the wild type and complementation strains. UDP-glucose may play an important role in increasing drug resistance to AMO, MNZ, CLA, LEV, TET, and RIF by maintaining the lipid A-core structure and decreasing membrane permeability. AlgC and GalU may serve as potential drug targets for decreasing antibiotic resistance in clinical isolates.

Metabolic Engineering of Escherichia coli for Efficient Biosynthesis of Lacto-N-tetraose Using a Novel β-1,3-Galactosyltransferase from Pseudogulbenkiania ferrooxidans.

Human milk oligosaccharides (HMOs) attract considerable interest in recent years because of their particular role in infant health. Lacto-N-tetraose (LNT), one of the most abundant HMOs, has been commercially added in the infant formula as a functional fortifier. In this study, a novel LNT-producing β-1,3-galactosyltransferase (β-1,3-GalT) from Pseudogulbenkiania ferrooxidans was screened from 14 putative candidates, and a highly LNT-producing metabolically engineered Escherichia coli strain was constructed based on a previously constructed lacto-N-triose II (LNT II)-producing strain, by strengthening UDP-galactose synthesis and introduction of P. ferrooxidans β-1,3-GalT. The engineered strain produced 3.11 and 25.49 g/L LNT in shake-flask and fed-batch cultivation, with the molar conversion ratio of LNT II to LNT of 88.15 and 85.09%, respectively. The productivity and specific yield of LNT in fed-batch cultivation were measured to be 0.61 g/L·h and 0.76 g/g dry cell weight, respectively. To the best of our knowledge, it is the highest LNT yield ever reported.

Efficient Production Hyperoside from Quercetin in Escherichia coli Through Increasing UDP-Galactose Supply and Recycling of Resting Cell

Hyperoside is used as the standard to assess the quality of Hyperin perforatum L and exhibits many biological properties. To improve the yield of hyperoside in Escherichia coli, four UDP-galactose synthesis pathways were screened and a recombinant strain was reconstructed by using an efficient UDP-galactose generation system coupled with Petunia hybrida glycosyltransferase to biosynthesis hyperoside from quercetin. By adopting the resting cell transformation, maximal hyperoside production of 4574 mg/L and average productivity of 63.5 mg/L/h were achieved after 72 h of biotranformation. Subsequently through recycling of resting cells, average productivity within 72 h reached 126 mg/L/h, which was 100% higher than that in the batch fermentation. Hyperoside production reached equivalent to 18,000 mg/L by recycling seven times of resting cell in 168 h, which was 393% of that in the batch fermentation and the first to reach 10 g per liter scale in E. coli. Therefore, this study provides an efficient method for construction of UDP-galactose synthesis pathway and hyperoside production in E. coli.

Designer biocatalysts for direct incorporation of exogenous galactose into globotriose.

Galactose is ubiquitous. The synthesis of galactose-containing oligosaccharides using Leloir galactosyltransferase requires uridine diphosphate (UDP)-galactose as the precursor. Of all UDP-galactose synthesis pathways developed for in vitro synthesis, the salvage pathway represents the simplest route. In this study, for the first time, we designed and constructed an Escherichia coli strain to use salvage pathway for UDP-galactose synthesis, demonstrating effective and direct incorporation of exogenous galactose into globotriose (Gb3). Successful establishment of salvage pathway enabled a complete delineation of carbon and energy source. Consequently, the designed biocatalyst was able to achieve high yield synthesis from galactose (0.95 moles of Gb3/moles galactose consumed) and a high product titer (2 g/L) in shaker flask within 24 hr. Elimination of limitation in acceptor sugar via homologous overexpression of LacY, the transporter for lactose, further improved the synthesis, raising Gb3 titer to 6 g/L in 24 hr and 7.5 g/L in 48 hr. The design principles successfully demonstrated in this study could be broadly applied for synthesis of other galactose-containing oligosaccharides. This study also illustrates a valid strategy to overcome limitation in the transport of acceptor sugar. As lactose is one of the most important basal structures, the significant improvement in synthesis through its enhanced transport could be emulated in numerous other lactose-based oligosaccharides.

Suppression of Alternative Lipooligosaccharide Glycosyltransferase Activity by UDP-Galactose Epimerase Enhances Murine Lung Infection and Evasion of Serum IgM.

In pathogens that produce lipooligosaccharide (LOS), sugar residues within the surface-exposed LOS outer core mediate interactions with components of the host immune system, promoting bacterial infection. Many LOS structures are controlled by phase variation mediated by random slipped-strand base mispairing, which can reversibly switch gene expression on or off. Phase variation diversifies the LOS, however its adaptive role is not well-understood. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen that causes a range of illnesses in the upper and lower respiratory tract. In NTHi a phase variable galactosyltransferase encoded by lic2A initiates galactose chain extension of the LOS outer core. The donor substrate for Lic2A, UDP-galactose, is generated from UDP-glucose by UDP-galactose epimerase encoded by galE. Our previous fitness profiling of H. influenzae mutants in a murine lung model showed that the galE mutant had a severe survival defect, while the lic2A mutant's defect was modest, leading us to postulate that unidentified factors act as suppressors of potential defects in a lic2A mutant. Herein we conducted a genome-wide genetic interaction screen to identify genes epistatic on lic2A for survival in the murine lung. An unexpected finding was that galE mutants exhibited restored virulence properties in a lic2A mutant background. We identified an alternative antibody epitope generated by Lic2A in the galE mutant that increased sensitivity to classical complement mediated killing in human serum. Deletion of lic2A or restoration of UDP-galactose synthesis alleviated the galE mutant's virulence defects. These studies indicate that when deprived of its galactosyl substrate, Lic2A acquires an alternative activity leading to increased recognition of NTHi by IgM and decreased survival in the lung model. Biofilm formation was increased by deletion of galE and by increased availability of UDP-GlcNAc precursors that can compete with UDP-galactose production. NTHi's ability to reversibly inactivate lic2A by phase-variation may influence survival in niches of infection in which UDP-Galactose levels are limiting.

UDP-Glucose 4-Epimerase and β-1,4-Galactosyltransferase from the Oyster Magallana gigas as Valuable Biocatalysts for the Production of Galactosylated Products.

Uridine diphosphate galactose (UDP-galactose) is a valuable building block in the enzymatic synthesis of galactose-containing glycoconjugates. UDP-glucose 4-epimerase (UGE) is an enzyme which catalyzes the reversible conversion of abundantly available UDP-glucose to UDP-galactose. Herein, we described the cloning, expression, purification, and biochemical characterization of an unstudied UGE from the oyster Magallana gigas (MgUGE). Activity tests of recombinantly expressed MgUGE, using HPLC (high-performance liquid chromatography), mass spectrometry, and photometric assays, showed an optimal temperature of 16 °C, and reasonable thermal stability up to 37 °C. No metal ions were required for enzymatic activity. The simple nickel-affinity-purification procedure makes MgUGE a valuable biocatalyst for the synthesis of UDP-galactose from UDP-glucose. The biosynthetic potential of MgUGE was further exemplified in a coupled enzymatic reaction with an oyster-derived β-1,4-galactosyltransferase (MgGalT7), allowing the galactosylation of the model substrate para-nitrophenol xylose (pNP-xylose) using UDP-glucose as the starting material.

Galactosylation of the Secondary Cell Wall Polysaccharide of Bacillus anthracis and Its Contribution to Anthrax Pathogenesis.

Bacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is essential for bacterial growth and cell division. B. anthracis SCWP is comprised of trisaccharide repeats with the structure, [→4)-β-ManNAc-(1→4)-β-GlcNAc(O3-α-Gal)-(1→6)-α-GlcNAc(O3-α-Gal, O4-β-Gal)-(1→]6-12 The genes whose products promote the galactosylation of B. anthracis SCWP are not yet known. We show here that the expression of galE1, encoding a UDP-glucose 4-epimerase necessary for the synthesis of UDP-galactose, is required for B. anthracis SCWP galactosylation. The galE1 mutant assembles surface (S) layer and S layer-associated proteins that associate with ketal-pyruvylated SCWP via their S layer homology domains similarly to wild-type B. anthracis, but the mutant displays a defect in γ-phage murein hydrolase binding to SCWP. Furthermore, deletion of galE1 diminishes the capsulation of B. anthracis with poly-d-γ-glutamic acid (PDGA) and causes a reduction in bacterial virulence. These data suggest that SCWP galactosylation is required for the physiologic assembly of the B. anthracis cell wall envelope and for the pathogenesis of anthrax disease.IMPORTANCE Unlike virulent Bacillus anthracis isolates, B. anthracis strain CDC684 synthesizes secondary cell wall polysaccharide (SCWP) trisaccharide repeats without galactosyl modification, exhibits diminished growth in vitro in broth cultures, and is severely attenuated in an animal model of anthrax. To examine whether SCWP galactosylation is a requirement for anthrax disease, we generated variants of B. anthracis strains Sterne 34F2 and Ames lacking UDP-glucose 4-epimerase by mutating the genes galE1 and galE2 We identified galE1 as necessary for SCWP galactosylation. Deletion of galE1 decreased the poly-d-γ-glutamic acid (PDGA) capsulation of the vegetative form of B. anthracis and increased the bacterial inoculum required to produce lethal disease in mice, indicating that SCWP galactosylation is indeed a determinant of anthrax disease.

Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization

Two different putative galactokinase genes, found in the genome database of Trypanosoma cruzi were cloned and sequenced. Expression of the genes in Escherichia coli resulted for TcGALK-1 in the synthesis of a soluble and active enzyme, and in the case of TcGALK-2 gene a less soluble protein, with predicted molecular masses of 51.9kDa and 51.3kDa, respectively. The Km values determined for the recombinant proteins were for galactose 0.108mM (TcGALK-1) and 0.091mM (TcGALK-2) and for ATP 0.36mM (TcGALK-1) and 0.1mM (TcGALK-2). Substrate inhibition by ATP (Ki 0.414mM) was only observed for TcGALK-2. Gel-filtration chromatography showed that natural TcGALKs and recombinant TcGALK-1 are monomeric. In agreement with the possession of a type-1 peroxisome-targeting signal by both TcGALKs, they were found to be present inside glycosomes using two different methods of subcellular fractionation in conjunction with mass spectrometry. Both genes are expressed in epimastigote and trypomastigote stages since the respective proteins were immunodetected by western blotting. The T. cruzi galactokinases present their highest (52–47%) sequence identity with their counterpart from Leishmania spp., followed by prokaryotic galactokinases such as those from E. coli and Lactococcus lactis (26–23%). In a phylogenetic analysis, the trypanosomatid galactokinases form a separate cluster, showing an affiliation with bacteria. Epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium supplemented with 20mM of galactose, suggesting that this hexose, upon phosphorylation by a TcGALK, could be used in the synthesis of UDP-galactose and also as a possible carbon and energy source.

Leishmania major UDP-sugar pyrophosphorylase salvages galactose for glycoconjugate biosynthesis

Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania whose severity ranges from self-healing cutaneous lesions to fatal visceral infections. Leishmania parasites synthesise a wide array of cell surface and secreted glycoconjugates that play important roles in infection. These glycoconjugates are particularly abundant in the promastigote form and known to be essential for establishment of infection in the insect midgut and effective transmission to the mammalian host. Since they are rich in galactose, their biosynthesis requires an ample supply of UDP-galactose. This nucleotide-sugar arises from epimerisation of UDP-glucose but also from an uncharacterised galactose salvage pathway. In this study, we evaluated the role of the newly characterised UDP-sugar pyrophosphorylase (USP) of Leishmania major in UDP-galactose biosynthesis. Upon deletion of the USP encoding gene, L. major lost the ability to synthesise UDP-galactose from galactose-1-phosphate but its ability to convert glucose-1-phosphate into UDP-glucose was fully maintained. Thus USP plays a role in UDP-galactose activation but does not significantly contribute to the de novo synthesis of UDP-glucose. Accordingly, USP was shown to be dispensable for growth and glycoconjugate biosynthesis under standard growth conditions. However, in a mutant seriously impaired in the de novo synthesis of UDP-galactose (due to deficiency of the UDP-glucose pyrophosphorylase) addition of extracellular galactose increased biosynthesis of the cell surface lipophosphoglycan. Thus under restrictive conditions, such as those encountered by Leishmania in its natural habitat, galactose salvage by USP may play a substantial role in biosynthesis of the UDP-galactose pool. We hypothesise that USP recycles galactose from the blood meal within the midgut of the insect for synthesis of the promastigote glycocalyx and thereby contributes to successful vector infection.

Ght2 + is required for UDP-galactose synthesis from extracellular galactose by Schizosaccharomyces pombe

Schizosaccharomyces pombe has eight hexose transporter genes, ght1 (+) to ght8 (+). Here we report that ght2 (+), which is highly expressed in the presence of glucose, is essential for UDP-galactose synthesis from extracellular galactose when cells grow on glucose. The galactosylation defect of a uge1Δ mutant defective in synthesis of UDP-galactose from glucose was suppressed in galactose-containing medium, but disruption of ght2 (+) in the uge1Δ mutant reversed suppression of the galactosylation defect. Expression of Saccharomyces cerevisiae GAL2 in uge1Δght2Δ cells suppressed the defective galactosylation phenotype in galactose-containing medium. These results indicate that galactose is transported from the medium to the cytosol in a Ght2-dependent manner, and is then converted into UDP-galactose.

ChemInform Abstract: Efficient One‐Pot Multienzyme Synthesis of UDP‐Sugars Using a Promiscuous UDP‐Sugar Pyrophosphorylase from Bifidobacterium longum (BLUSP).

A promiscuous UDP-sugar pyrophosphorylase (BLUSP) was cloned from Bifidobacterium longum strain ATCC55813 and used efficiently with a Pasteurella multocida inorganic pyrophosphatase (PmPpA) with or without a monosaccharide 1-kinase for one-pot multienzyme synthesis of UDP-galactose, UDP-glucose, UDP-mannose, and their derivatives. Further chemical diversification of a UDP-mannose derivative resulted in the formation of UDP-N-acetylmannosamine.

Gene regulation of UDP-galactose synthesis and transport: potential rate-limiting processes in initiation of milk production in humans

Lactose synthesis is believed to be rate limiting for milk production. However, understanding the molecular events controlling lactose synthesis in humans is still rudimentary. We have utilized our established model of the RNA isolated from breast milk fat globule from seven healthy, exclusively breastfeeding women from 6 h to 42 days following delivery to determine the temporal coordination of changes in gene expression in the carbohydrate metabolic processes emphasizing the lactose synthesis pathway in human mammary epithelial cell. We showed that milk lactose concentrations increased from 75 to 200 mM from 6 to 96 h. Milk progesterone concentrations fell by 65% at 24 h and were undetectable by day 3. Milk prolactin peaked at 36 h and then declined progressively afterward. In concordance with lactose synthesis, gene expression of galactose kinase 2, UDP-glucose pyrophosphorylase 2 (UGP2), and phosphoglucomutase 1 increased 18-, 10-, and threefold, respectively, between 6 and 72 h. Between 6 and 96 h, gene expression of UDP-galactose transporter 2 (SLC35A2) increased threefold, whereas glucose transporter 1 was unchanged. Gene expression of lactose synthase no. 3 increased 1.7-fold by 96 h, whereas α-lactalbumin did not change over the entire study duration. Gene expression of prolactin receptor (PRLR) and its downstream signal transducer and activator of transcription complex 5 (STAT5) were increased 10- and 2.5-fold, respectively, by 72 h. In summary, lactose synthesis paralleled the induction of gene expression of proteins involved in UDP-galactose synthesis and transport, suggesting that they are potentially rate limiting in lactose synthesis and thus milk production. Progesterone withdrawal may be the signal that triggers PRLR signaling via STAT5, which may in turn induce UGP2 and SLC35A2 expression.

Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP)

A promiscuous UDP-sugar pyrophosphorylase (BLUSP) was cloned from Bifidobacterium longum strain ATCC55813 and used efficiently with a Pasteurella multocida inorganic pyrophosphatase (PmPpA) with or without a monosaccharide 1-kinase for one-pot multienzyme synthesis of UDP-galactose, UDP-glucose, UDP-mannose, and their derivatives. Further chemical diversification of a UDP-mannose derivative resulted in the formation of UDP-N-acetylmannosamine.

Structure and Function of the d -Galactose Network in Enterobacteria

ABSTRACTGalactose is important for the survival and virulence of bacteria. In Escherichia coli, galactose is utilized by the Leloir pathway, which is controlled by a complex network. To shed light on the potential functions the galactose network could perform, we performed bioinformatical analysis of reference genome sequences belonging to the Enterobacteriaceae family. We found that several genomes have reduced numbers of components compared to the E. coli galactose system, suggesting that the network can be optimized for different environments. Typically, genes are removed by deletions; however, in Yersinia pestis, the galactose mutarotase (galM) gene is inactivated by a single-base-pair deletion. Lack of GalM activity indicates that the two anomers of d-galactose are used for different purposes, α-d-galactose as a carbon source and β-d-galactose for induction of UDP-galactose synthesis for biosynthetic glycosylation. We demonstrate that activity of the galM gene can be restored by different single-base-pair insertions. During the evolution of Y. pestis to become a vector-transmitted systemic pathogen, many genes were converted to pseudogenes. It is not clear whether pseudogenes are present to maintain meiotrophism or are in the process of elimination. Our results suggest that the galM pseudogene has not been deleted because its reactivation may be beneficial in certain environments.

Sorting of phosphoglucomutase to glycosomes in Trypanosoma cruzi is mediated by an internal domain

Trypanosoma cruzi relies on highly galactosylated molecules as virulence factors and the enzymes involved in sugar biosynthesis are potential therapeutic targets. The synthesis of UDP-galactose in T. cruzi requires the activity of phosphoglucomutase (PGM), the enzyme that catalyzes the interconversion of glucose-6-phosphate and glucose-1-phosphate. Several enzymes that participate in carbohydrate metabolism in trypanosomes are confined to specialized peroxisome-like organelles called glycosomes. The majority of glycosomal proteins contain peroxisome-targeting signals (PTS) at the COOH- or at the amino-terminus, which drive their transport to glycosomes. We had previously identified the T. cruzi PGM gene (TcPGM) and demonstrated that it encodes a functional enzyme. Here, we show that, in contrast to yeast and mammalian cells, TcPGM resides in glycosomes of the parasite. However, no classical PTS1 or PTS2 motif is present in its sequence. We investigated glycosomal targeting by generating T. cruzi cell lines expressing different domains of TcPGM fused to the green fluorescent protein (GFP). The analysis of the subcellular localization of fusion proteins revealed that an internal targeting signal of TcPGM, residing between amino acid residues 260 and 380, is capable of targeting GFP to glycosomes. These results demonstrate that, in T. cruzi, PGM import into glycosomes is mediated by a novel non-PTS domain that is located internally in the protein.

Open and Closed Structures of the UDP-glucose Pyrophosphorylase from Leishmania major

Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel beta-helix (LbetaH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.

Conversion of Capsular Polysaccharide, Involved in Pellicle Formation, to Extracellular Polysaccharide bygalEDeletion inAcetobacter tropicalis

Acetobacter tropicalis SKU1100 produces a pellicle-forming capsular polysaccharide (CPS), consisting of galactose, glucose, and rhamnose. We cloned the galE gene, a UDP-galactose synthesis gene, from A. tropicalis SKU1100 by PCR. A galE-disruptant was prepared and found not to produce CPS and thus not to form a pellicle under the static condition. Instead, the DeltagalE mutant secreted an extracellular polysaccharide (EPS), which was purified and found to have a unique character, different from the original CPS.