The molecular architecture of glucose-1-phosphate uridylyltransferase

Glucose-1-phosphate uridylyltransferase, also referred to as UDP-glucose pyrophosphorylase or UGPase, catalyzes the formation of UDP-glucose from glucose-1-phosphate and UTP. Not surprisingly, given the central role of UDP-glucose in glycogen synthesis and in the production of glycolipids, glycoproteins, and proteoglycans, the enzyme is ubiquitous in nature. Interestingly, however, the prokaryotic and eukaryotic forms of the enzyme are unrelated in amino acid sequence and structure. Here we describe the cloning and structural analysis to 1.9 A resolution of the UGPase from Escherichia coli. The protein is a tetramer with 222 point group symmetry. Each subunit of the tetramer is dominated by an eight-stranded mixed beta-sheet. There are two additional layers of beta-sheet (two and three strands) and 10 alpha-helices. The overall fold of the molecule is remarkably similar to that observed for glucose-1-phosphate thymidylyltransferase in complex with its product, dTDP-glucose. On the basis of this similarity, a UDP-glucose moiety has been positioned into the active site of UGPase. This protein/product model predicts that the side chains of Gln 109 and Asp 137, respectively, serve to anchor the uracil ring and the ribose of UDP-glucose to the protein. The beta-phosphoryl group of the product is predicted to lie within hydrogen bonding distance to the epsilon-nitrogen of Lys 202 whereas the carboxylate group of Glu 201 is predicted to bridge the 2'- and 3'-hydroxyl groups of the glucosyl moiety. Details concerning the overall structure of UGPase and a comparison with glucose-1-phosphate thymidylyltransferase are presented.     

Conversion of glucose-1-phosphate to 3-keto-glucose-1-phosphate by cells of Agrobacterium tumefaciens

Incubation of resting cells of Agrobacterium tumefaciens with glucose-1-phosphate resulted in the accumulation of a new sugar phosphate in the suspending medium. Approximately 80% of the glucose-1-phosphate consumed was converted to the new compound, which was identified as alpha-d-ribo-hexopyranosyl-3-ulose-1-phosphate (3-ketoglucose-1-phosphate). Both utilization of glucose-1-phosphate and accumulation of 3-ketoglucose-1-phosphate were inhibited by 2,4-dinitrophenol, polymyxin, and d-glucose, which are inhibitors of the glucoside transport system of this bacterium but are not inhibitors of d-glucoside-3-dehydrogenase, which is the 3-ketoglucose-1-phosphate-forming enzyme. Consequently, it was concluded that glucose-1-phosphate penetrates into intracellular space by means of an active transport system. The glucose-1-phosphate is converted to 3-ketoglucose-1-phosphate by d-glucoside-3-dehydrogenase, and the 3-ketoglucose-1-phosphate formed reaches the extracellular space by passing through the surface layer of the bacterium.     

Cloning and characterization of CalS7 from Micromonospora echinospora sp. calichensis as a glucose-1-phosphate nucleotidyltransferase

The deoxysugar biosynthetic gene cluster of calicheamicin contains the calS7, which encodes glucose-1-phosphate nucleotidyltransferase and converts glucose-1-phosphate and nucleotides (NTP) to NDP-glucose and pyrophosphate. calS7 was expressed in Escherichia coli BL21(DE3), and the purified protein had significant thymidylyltransferase and uridylyltransferase activities as well, with some guanidylyltransferase activity but negligible cytidyl and adenyltransferase activity. The functions of thymidylyltransferase and uridylyltransferase were also verified using one-pot enzymatic synthesis of TMK and ACK. The products were analyzed by HPLC and ESI/MS, which showed peaks at m/z = 563 and 565 for TDP-d-glucose and UDP-d-glucose, respectively, in negative mode.     

Active site geometry of glucose-1-phosphate uridylyltransferase

Glucose-1-phosphate uridylyltransferase, or UGPase, catalyzes the production of UDP-glucose from glucose-1-phosphate and UTP. Because of the biological role of UDP-glucose in glycogen synthesis and in the formation of glycolipids, glycoproteins, and proteoglycans, the enzyme is widespread in nature. Recently this laboratory reported the three-dimensional structure of UGPase from Escherichia coli. While the initial X-ray analysis revealed the overall fold of the enzyme, details concerning its active site geometry were limited because crystals of the protein complexed with either substrates or products could never be obtained. In an effort to more fully investigate the active site geometry of the enzyme, UGPase from Corynebacterium glutamicum was subsequently cloned and purified. Here we report the X-ray structure of UGPase crystallized in the presence of both magnesium and UDP-glucose. Residues involved in anchoring the ligand to the active site include the polypeptide chain backbone atoms of Ala 20, Gly 21, Gly 117, Gly 180, and Ala 214, and the side chains of Glu 36, Gln 112, Asp 143, Glu 201, and Lys 202. Two magnesium ions are observed coordinated to the UDP-glucose. An alpha- and a beta-phosphoryl oxygen, three waters, and the side chain of Asp 142 ligate the first magnesium, whereas the second ion is coordinated by an alpha-phosphoryl oxygen and five waters. The position of the first magnesium is conserved in both the glucose-1-phosphate thymidylyltransferases and the cytidylyltransferases. The structure presented here provides further support for the role of the conserved magnesium ion in the catalytic mechanisms of the sugar-1-phosphate nucleotidylyltransferases.     

Active transport of glucose-1-phosphate in Agrobacterium tumefaciens

The presence of an active transport system for glucose-1-phosphate in Agrobacterium tumefaciens was demonstrated from the following observations. (i) The bacterium could grow on a medium containing glucose-1-phosphate as carbon source; (ii) the entry of glucose-1-phosphate into the resting cells occurred against concentration gradient obeying Michaelis-Menten kinetics; and (iii) the entry reaction was energy-dependent. The transport system for glucose-1-phosphate was formed inducibly by growing the organism on a glucose-1-phosphate or sucrose medium. From the inhibition and kinetics studies it was found that the transport system had a high specificity for glucose-1-phosphate with a high affinity, K(m) value of 4.5 x 10(-6)m at pH 8.2. The existence of glucose-1-phosphate binding factor was proved in the shock fluid which was prepared from the cells grown on both glucose-1-phosphate and sucrose media by osmotic shock.     

One-pot enzymatic synthesis of UDP-D-glucose from UMP and glucose-1-phosphate using an ATP regeneration system

Glucose-1-phosphate uridylyltransferase from E. coli K12 was used to convert uridine-5'-triphosphate and glucose-1-phosphate to UDP-D-glucose. The conversion was efficient and completed within 5 minutes under the employed conditions. In addition, thymidine-5'-monophosphate kinase and acetate kinase were proven to be non-specific, converting udridine-5'-monophosphate to uridine-5'-triphosphate with 55% conversion after 6 h, which was much slower than the production of TTP under the same conditions (complete conversion within one hour). Since these two reactions could proceed under the same conditions, a one-pot synthesis of UDP-D-glucose with ATP regeneration was designed from easily available starting materials, and conversion up to 40% by HPLC peak integration was achieved given a reaction time of 4 h.     

Evidence for glucose-6-phosphate transport in rat liver microsomes

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.     

Hyperanodic forms of human glucose-6-phosphate dehydrogenase.

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into 'hyperanodic forms' when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some 'NADP+ modifying proteins' purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.     

Proteins encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG genes, involved in gellan gum biosynthesis, exhibit both dTDP- and UDP-glucose pyrophosphorylase activities

The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Km of 12.0 microM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Km of 229.0 microM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).     

Development of a glucose-6-phosphate biosensor based on coimmobilized p-hydroxybenzoate hydroxylase and glucose-6-phosphate dehydrogenase

This work reports the development of an amperometric glucose-6-phosphate biosensor by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and glucose-6-phosphate dehydrogenase (G6PDH) on a screen-printed electrode. The principle of the determination scheme is as follows: G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NADP(+). The product, NADPH, initiates the irreversible the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a detectable signal due to its oxidation at the working electrode. The sensor shows a broad linear detection range between 2 microM and 1000 microM with a low detection limit of 1.2 microM. Also, it has a fast measuring time which can achieve 95% of the maximum current response in 20s after the addition of a given concentration of glucose-6-phosphate with a short recovery time (2 min).     

Mutarotase (aldose-1-epimerase) catalyzed anomerization of glucose-6-phosphate

Data obtained by direct polarimetric analysis show that glucose-6-phosphate is a mutarotase (aldose-1-epimerase) substrate; that the enzyme is most active against glucose-6-phosphate at slightly acid pH; and that the monoanion form of glucose-6-phosphate is probably the form involved with mutarotase.     

The roles of galactitol, galactose-1-phosphate, and phosphoglucomutase in galactose-induced toxicity in Saccharomyces cerevisiae

The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combinations of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furthermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.     

Characterization of the <Emphasis Type="Italic"> ugpG </Emphasis>gene encoding a UDP-glucose pyrophosphorylase from the gellan gum producer <Emphasis Type="Italic"> Sphingomonas paucimobilis </Emphasis>ATCC 31461

The ugpGgene, which codes for a UDP-glucose pyrophosphorylase (UGP) (or glucose-1-phosphate uridylyltransferase; EC 2.7.7.9) in Sphingomonas paucimobilis ATCC 31461, was cloned and sequenced. This industrial strain produces the exopolysaccharide gellan, a new commercial gelling agent, and the ugpG gene may convert glucose-1-phosphate into UDP-glucose in the gellan biosynthetic pathway. The ugpG gene is capable of restoring the capacity of an Escherichia coli galU mutant to grow on galactose by functional complementation of its deficiency for UDP-glucose pyrophosphorylase activity. As expected, the predicted gene product shows strong homology to UDP-glucose pyrophosphorylases from several bacterial species. The N-terminal region of UgpG exhibits the motif GXGTRXLPXTK, which is highly conserved among bacterial XDP-sugar pyrophosphorylases, and a lysine residue (K(192)) is located within a VEKP motif predicted to be essential for substrate binding or catalysis. UgpG was purified to homogeneity as a heterologous fusion protein from crude cell extracts prepared from IPTG-induced cells of E. coli, using affinity chromatography. Under denaturing conditions, the fusion protein S-UgpG-His(6) migrated with an estimated molecular mass of 36 kDa [corresponding to the predicted molecular mass of native UgpG (31.2 kDa) plus 5 kDa for the S and histidine tags). Kinetic analysis of UgpG in the reverse reaction (pyrophosphorolysis) showed a typical Michaelis-Menten substrate saturation pattern. The apparent K(m) and V(max) values estimated for UDP-glucose were 7.5 microM and 1275 micromol/min/g.     

Genome sequence of Sphingomonas elodea ATCC 31461, a highly productive industrial strain of gellan gum

The commercial gelling agent gellan gum is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. However, the genes involved in the biosynthesis, regulation, and modification of gellan gum have not been fully characterized. Here we describe the draft genome sequence of stain ATCC 31461 and major findings from its annotation.     

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of Escherichia coli glucose-1-phosphate thymidylyltransferase (RffH) complexed with dTTP and Mg2+

The enzyme glucose-1-phosphate thymidylyltransferase (RffH), the product of the rffh gene, catalyzes one of the steps in the synthesis of enterobacterial common antigen (ECA), a cell surface glycolipid found in Gram-negative enteric bacteria. In Escherichia coli two gene products, RffH and RmlA, catalyze the same enzymatic reaction and are homologous in sequence; however, they are part of different operons and function in different pathways. We report the crystal structure of RffH bound to deoxythymidine triphosphate (dTTP), the phosphate donor, and Mg(2+), refined at 2.6 A to an R-factor of 22.3% (R(free) = 28.4%). The crystal structure of RffH shows a tetrameric enzyme best described as a dimer of dimers. Each monomer has an overall alpha/beta fold and consists of two domains, a larger nucleotide binding domain (residues 1-115, 222-291) and a smaller sugar-binding domain (116-221), with the active site located at the domain interface. The Mg(2+) ion is coordinated by two conserved aspartates and the alpha-phosphate of deoxythymidine triphosphate. Its location corresponds well to that in a structurally similar domain of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). Analysis of the RffH, RmlA, and GlmU complexes with substrates and products provides an explanation for their different affinities for Mg(2+) and leads to a proposal for the dynamics along the reaction pathway.     

Eukaryotic glucose-6-phosphate dehydrogenases: Structural screening of related proteins

Rapid assessment of structural relationships between yeast glucose-6-phosphate dehydrogenases and other eukaryotic types of this enzyme is described. Separation and size estimation of large fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis, electroblotting onto disks, and sequencer analysis provide data that permit alignment of the segments thus characterized with the related proteins, and utilize existing structural knowledge to assess new enzyme structures. Affinity labeling allows further correlations. The results establish the overall structural arrangements of the new proteins, including the location of the active-site lysine residue, even though the yeast enzyme structures are found to differ markedly from the few previously characterized glucose-6-phosphate dehydrogenases.     

Continuous production of glucose-6-phosphate by immobilized Achromobacter butyri cells

Summary Whole cells of Achromobacter butyri OUT 8004 having polyphosphate glucokinase activity were immobilized in polyacrylamide gel. The immobilized cells were activated by organic solvents, especially acetone. The immobilization resulted in increased stability of polyphosphate glucokinase. Continuous high yield production of G-6-P from glucose and metaphosphate was performed with an immobilized cell column, which had a half-life of approximately 20 days.Abbreviations G-6-P glucose-6-phosphate - G-1-P glucose-1-phosphate - Cation-S stearyl trimethyl ammonium chloride - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)-aminomethane; p-NPP, p-nitrophenyl phosphate - S.V. space velocity     

Kinetic and crystallographic analyses support a sequential-ordered bi bi catalytic mechanism for Escherichia coli glucose-1-phosphate thymidylyltransferase.

Glucose-1-phosphate thymidylyltransferase is the first enzyme in the biosynthesis of dTDP-l-rhamnose, the precursor of l-rhamnose, an essential component of surface antigens, such as the O-lipopolysaccharide, mediating virulence and adhesion to host tissues in many microorganisms. The enzyme catalyses the formation of dTDP-glucose, from dTTP and glucose 1-phosphate, as well as its pyrophosphorolysis. To shed more light on the catalytic properties of glucose-1-phosphate thymidylyltransferase from Escherichia coli, specifically distinguishing between ping pong and sequential ordered bi bi reaction mechanisms, the enzyme kinetic properties have been analysed in the presence of different substrates and inhibitors. Moreover, three different complexes of glucose-1-phosphate thymidylyltransferase (co-crystallized with dTDP, with dTMP and glucose-1-phosphate, with d-thymidine and glucose-1-phosphate) have been analysed by X-ray crystallography, in the 1.9-2.3 A resolution range (R-factors of 17.3-17.5 %). The homotetrameric enzyme shows strongly conserved substrate/inhibitor binding modes in a surface cavity next to the topological switch-point of a quasi-Rossmann fold. Inspection of the subunit tertiary structure reveals relationships to other enzymes involved in the biosynthesis of nucleotide-sugars, including distant proteins such as the molybdenum cofactor biosynthesis protein MobA. The precise location of the substrate relative to putative reactive residues in the catalytic center suggests that, in keeping with the results of the kinetic measurements, both catalysed reactions, i.e. dTDP-glucose biosynthesis and pyrophosphorolysis, follow a sequential ordered bi bi catalytic mechanism.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.