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Background As the main form of photoassimilates transported from vegetative tissues to the reproductive organs, sucrose and its degradation products are crucial for cell fate determination and development of maize kernels. Despite the relevance of sucrose synthase SH1 (shrunken 1)-mediated release of hexoses for kernel development, the underlying physiological and molecular mechanisms are not yet well understood in maize (Zea mays). Results Here, we identified a new allelic mutant of SH1 generated by EMS mutagenesis, designated as sh1*. The mutation of SH1 caused more than 90% loss of sucrose synthase activity in sh1* endosperm, which resulted in a significant reduction in starch contents while a dramatic increase in soluble sugars. As a result, an extremely high osmolality in endosperm cells of sh1* was generated, which caused kernel swelling and affected the seed development. Quantitative measurement of phosphorylated sugars showed that Glc-1-P in endosperm of sh1* (17 μg g− 1 FW) was only 5.2% of that of wild-type (326 μg g− 1 FW). As a direct source of starch synthesis, the decrease of Glc-1-P may cause a significant reduction in carbohydrates that flow to starch synthesis, ultimately contributing to the defects in starch granule development and reduction of starch content. Conclusions Our results demonstrated that SH1-mediated sucrose degradation is critical for maize kernel development and starch synthesis by regulating the flow of carbohydrates and maintaining the balance of osmotic potential.

Sucrose phosphorylases are carbohydrate-active enzymes with outstanding potential forthe biocatalytic conversion of common table sugar into products with attractive properties. Theybelong to the glycoside hydrolase family GH13, where they are found in subfamily 18. In bacteria,these enzymes catalyse the phosphorolysis of sucrose to yield alpha-glucose 1-phosphate and fructose. However, sucrose phosphorylases can also be applied as versatile transglucosylases for the synthesisof valuable glycosides and sugars because their broad promiscuity allows them to transfer theglucosyl group of sucrose to a diverse collection of compounds other than phosphate. Numerousprocess and enzyme engineering studies have expanded the range of possible applications of sucrosephosphorylases ever further. Moreover, it has recently been discovered that family GH13 also containsa few novel phosphorylases that are specialised in the phosphorolysis of sucrose 6F-phosphate,glucosylglycerol or glucosylglycerate. In this review, we provide an overview of the progress that hasbeen made in our understanding and exploitation of sucrose phosphorylases and related enzymesover the past ten years.

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.

In biotechnological or other applications, electrodialysis can be an efficient method for the separation of phosphate from products with relatively high molecular masses (greater than around 200 kg/kmol), even though they are electrically charged. In this work, the separation of phosphate at high initial concentration (360 mM) from glucose-1-phosphate (G-1-P, 46 mM) was investigated. Around 90% of the phosphate could be removed, while 25% of G-1-P was separated. In this case, there is no product loss because phosphate could be recycled back to the reactor. Also at low concentrations (CP ∼ 22 mM), 77% of phosphate and only 20% of G-1-P, (CG-1-P ∼ 30 mM) were removed. The efficiency can be substantially improved with a two-step electrodialysis. The molar phosphate fluxes were considerably higher than the G-1-P (from around 4 to 40 times) at phosphate to G-1-P concentration ratios from 0.7 to 7. A theoretical consideration shows that this can be first of all attributed to a rather low mobility of G-1-P in the membrane matrix. The mobility of such substances in ion-exchange membranes depends also significantly on concentration.

α-Glucose 1-phosphate (G1P) is synthesized from 5% (w/v) corn starch and 1 M phosphate mediated by α-glucan phosphorylase (αGP) from the thermophilic bacterium Thermotoga maritima at pH 7.2 and 70 °C. To increase G1P yield from corn starch containing branched amylopectin, a hyper-thermostable isoamylase from Sulfolobus tokodaii was added for simultaneous starch gelatinization and starch-debranching hydrolysis at 85 °C and pH 5.5 before αGP use. The pretreatment of isoamylase increased G1P titer from 120 mM to 170 mM. To increase maltose and maltotriose utilization, the third thermostable enzyme, 4-glucanotransferase (4GT) from Thermococcus litoralis, was added during the late stage of G1P biotransformation, further increasing G1P titer to 200 mM. This titer is the highest G1P level obtained on starch or its derived products (maltodextrin and soluble starch). This study suggests that in vitro multienzyme biotransformation has an advantage of great engineering flexibility in terms of space and time compared with microbial fermentation.

UDP-glucose pyrophosphorylase (UGP) is a key enzyme involved in carbohydrate metabolism, but there are few studies on the functions of this enzyme in fungi. The ugp gene of Ganoderma lucidum was cloned, and enzyme kinetic parameters of the UGP recombinant protein were determined in vitro, revealing that this protein was functional and catalyzed the reversible conversion between Glc-1-P and UDP-Glc. ugp silencing by RNA interference resulted in changes in the levels of the intermediate metabolites Glc-1-P and UDP-Glc. The compounds and structure of the cell wall in the silenced strains were also altered compared with those in the wild-type strains. Moreover, the number of hyphal branches was also changed in the silenced strains. To verify the role of UGP in hyphal branching, a ugp-overexpressing strain was constructed. The results showed that the number of hyphal branches was influenced by UGP. The mechanism underlying hyphal branching was further investigated by adding exogenous Glc-1-P. Our results showed that hyphal branching was regulated by a change in the cytosolic Ca(2+) concentration, which was affected by the level of the intermediate metabolite Glc-1-P, in G. lucidum. Our findings indicate the existence of an interaction between carbon metabolism and Ca(2+) signaling in this fungus.

Starch phosphorylase is a α-glucan-phosphorylase that plays various important roles in the regulation of carbohydrate metabolism. It is mainly involved in the degradation of starch, however, it has also been reported to be involved in the synthesis of starch especially during abiotic stress conditions. Mutant studies revealed that disruption of this enzyme adversely affects the survival of plants. In the present study, we have established that starch phosphorylase is highly conserved among plant species in terms of protein sequence and 3-dimensional structure. Besides, we theorized degradation by starch phosphorylase as a common plausible mechanism through which herbicides may exert adverse effects on general plant population. Using in silico biology, we provided a clue that some of the commercially available herbicides have potential to bind with the active site of starch phosphorylase and these can competitively inhibit the activity of this vital enzyme.

Starch phosphorylase (EC 2.4.1.1) is the key enzyme which catalyzes the reversible conversion of starch and inorganic phosphate into glucose-1-phosphate (G-1-P) and has been reported in many higher plants. It can be exploited for industrial purposes for tailoring starch and for the synthesis of biopolymers which may be useful in food industry and are also of clinical importance. Starch in the form of insoluble granules accumulates in chloroplasts as the primary product of photosynthesis. Multiple forms of the enzyme have been reported in different plant tissues which have been predicted to have different roles in starch metabolism. Here, various biochemical properties have Review Article Jain et al.; BBJ, 5(3): 103-122, 2015; Article no.BBJ.2015.011 104 been reviewed. Various techniques for enzyme immobilization have been discussed. Besides, reports on immobilization of starch phosphorylase from different sources and on different solid matrices have also been reviewed. Peptide mapping reports of various proteins have also been assessed in this review.

Background The biosynthesis of NDP-glucoses is based on the nucleotide transfer from NTP donor substrates to glucose-1-phosphates catalyzed by glucose-1-phosphate nucleotidyltransferases. Objectives The cloning and biochemical characterization of a glucose-1-phosphate nucleotidyltransferase (TiGPNT) from the deep sea bacterium Thermodesulfatator indicus. Methods The biochemical parameters of recombinant TiGPNT were determined using a plate reader-based coupled enzymatic assay, in which the reaction product UDP-glucose is oxidized in the presence of NAD+ forming UDP-Glucuronic acid and NADH. The substrate promiscuity of the enzyme was determined using thin-layer chromatography and MALDI-ToF mass spectrometry. Results TiGPNT was recombinantly expressed under the control of the T7 promoter in Escherichia coli and could be successfully enriched by heat treatment at 80°C for 30 min. The obtained enzyme worked best at pH 7.5 and the optimum reaction temperature was determined to be 50°C. Interestingly, TiGPNT could fully retain its activity even after extended incubation periods at temperatures of up to 80°C. The enzyme was strongly inhibited in the presence of Cu2+ and Fe2+ ions and EDTA. Among the tested glycosyl donor substrates, TiGPNT showed strict specificity towards glucose-1-phosphate. At the same time, TiGPNT was highly promiscuous towards all tested nucleotide donor substrates. Conclusion TiGPNT shows comparable biochemical features in regards to pH optima, temperature optima and the substrate specificity to characterized glucose-1-phosphate nucleotidyltransferase from other species. The enzyme was capable of utilizing glucose-1-phosphate and all tested nucleoside triphosphate donors as substrates. The high activity of the enzyme and the simple purification protocol make TiGPNT an interesting new biocatalyst for the synthesis of glucose-diphospho nucleosides.

Cellobiose phosphorylase (EC 2.4.1.20, CBP) catalyzes the reversible phosphorolysis of cellobiose to alpha-D-glucose 1-phosphate (Glc1P) and D-glucose. Cys485, Tyr648, and Glu653 of CBP from Ruminococcus albus, situated at the +1 subsite, were mutated to modulate acceptor specificity. C485A, Y648F, and Y648V were active enough for analysis. Their acceptor specificities were compared with the wild type based on the apparent kinetic parameters determined in the presence of 10 mM Glc1P. C485A showed higher preference for D-glucosamine than the wild type. Apparent k(cat)/K-m values of Y648F for D-mannose and 2-deoxy-D-glucose were 8.2- and 4.0-fold higher than those of the wild type, respectively. Y648V had synthetic activity toward N-acetyl-D-glucosamine, while the other variants did not. The oligosaccharide production in the presence of the same concentrations of wild type and each mutant was compared. C485A produced 4-O-beta-D-glucopyranosyl-D-glucosamine from 10 mM Glc1P and D-glucosamine at a rate similar to the wild type. Y648F and Y648V produced 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine much more rapidly than the wild type when D-mannose and N-acetyl-D-glucosamine were used as acceptors, respectively. After a 4 h reaction, the amounts of 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine produced by Y648F and Y648V were 5.9- and 12-fold higher than the wild type, respectively. (C) 2013 Elsevier Ltd. All rights reserved.

Synthetic methods were investigated for the preparation of O and S -glucosyl thiophosphates and glucosyl 1C-thiophosphonate. Four protected glucosyl thiophosphate compounds were synthesized and characterized as precursors to glucose 1-thiophosphate. The effect of various reaction conditions and the nature of the carbohydrate and thiophosphate protecting groups and how they impact both the yields and α/β diastereoselectivity of the glucosyl thiophosphate products were explored. A novel isomerization from an O -linked to S -linked glucosyl thiophosphate was observed. α- d -Glucose-1C-thiophosphonate was synthesized and evaluated as a substrate for the thymidylyltransferase, Cps2L. Tandem mass spectrometric analysis determined the position of sulfur in the sugar nucleotide product.

Starch phosphorhylase extracted from mature potato tubers (Solanum tuberosum L. var. Kufri Badshah) was partially purified and immobilized on brick dust as solid support via glutaraldehyde. The percentage retention of the enzyme activity on brick dust was nearly 80 %. After immobilization specific activity of the enzyme increased from 0.92 to 3.25 U mg−1 protein with about 3.5-fold enrichment. The optimum pH and temperature of soluble enzyme were determined as pH 6.0 and 37 °C, respectively whereas immobilized enzyme showed at pH 6.5 and 47 °C, respectively. The immobilized enzyme displayed higher thermal stability than soluble enzyme and retained about 50 % of its initial activity after 10 reuses. Immobilized enzyme was packed in an indigenously designed double walled glass bed reactor for continuous production of glucose-1-phosphate.

The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-C-14]glucose 1-phosphate, [U-C-14]sucrose, [U-C-14]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-C-14]sucrose plus unlabelled equimolar glucose 1-phosphate. C-14-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced C-14 incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 degrees C but the flux of the sucrose-dependent route strongly increases above 20 degrees C. Results are confirmed by in vitro experiments using [U-C-14]glucose 1-phosphate or adenosine-[U-C-14]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C-14-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.

Sucrose phosphorylase (SP) is a useful biocatalyst for the selective transfer of α-glucosyl residues to a variety of acceptor molecules. Its industrial application is, however, hampered by the lack of enzyme variants that can withstand the process temperature of 60 °C. We have recently shown that the stability of the SP from Bifidobacterium adolescentis can be improved by immobilization on Sepabeads EC-HFA, and have now applied this biocatalyst for the continuous production of α- d -glucose-1-phosphate from sucrose. To lower the costs, the enzyme has only been partially purified prior to immobilization. Interestingly, the presence of substrate was found to dramatically enhance the stability of the biocatalyst, allowing its use in a packed-bed reactor for more than 2 weeks at 60 °C without loss of activity. The overall process generated a space-time yield of 179 g/l/h, and the product could be recovered in crystalline form with a yield of 86%.

Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [C-14] glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.

The continuous production of Glucose-1-phosphate (G-1-P) using immobilized potato phosphorylase was investigated. Potato phosphorylase was immobilized on a strongly basic anion-exchange resin, HPA-25 through contact of the resin with potato juice or wastewater of a potato-starch factory. The immobilization was highly selective for phosphorylase among proteins contained in potato juice and the wastewater. The immobilized phosphorylase could be used for the continuous G-1-P production over one year without significant deactivation. By using a 100-L column reactor packed with immobilized phosphorylase of 14.1 U·mL-carrier–1 at the superficial linear velocity of 0.034 m·h–1 and the space velocity of 3.1 × 10–9 m3·U–1·h–1, 185 mM G-1-P, which is the maximum concentration potentially expected from 516 mM dextrin and 2000 mM phosphate, was continuously produced.

Starch phosphorylase has been isolated from cabbage (Elephantopus scabar) leaves and partially purified using ammonium sulfate fractionation. The partially purified enzyme was desalted using Sephadex-G-25 chromatography. In the direction of polysaccharide synthesis, the enzyme showed optimum activity at pH 6.0 with two half pH optima at pH 5.3 and pH 7.1 whereas in the direction of glucose-1-phosphate formation, it showed optimum pH at pH 7.0 with half pH-optima at pH 6.4 and 7.6. The optimum temperature for the enzyme activity has been found to be 37ºC with two half temperature optima at 34ºC and 40ºC. The partially purified enzyme has been immobilized using egg shell as solid support. The percentage retention of the enzyme on egg shell was nearly 56%. After immobilization, specific activity of the enzyme increased from 0. 0225 to 0.0452. Upon immobilization, there was a slight alkaline shift in the optimum pH when assayed in both the directions. The immobilized enzyme also displayed increased optimum temperature and thermo-stability and could be reused number of times. The increase in thermo-stability and reusability of the immobilized enzyme has been exploited for the production of glucose-1-phosphate, a cytostatic compound used in cardio-therapy. The glucose-1-phosphate produced has been purified with nearly 95% purity after adsorption chromatography on norite and ion exchange chromatography on DEAE cellulose.

Glucose-1-phosphate (G-1-P) is a biological product which, along with its derivatives, has various physiological applications. In order to develop an efficient industrial process for G-1-P, enzymatic production of high concentration G-1-P was attempted by using phosphorylase purified from potatoes as an enzyme, several kinds of linear and branched dextrin differing in chain length and a mixture of KH2PO4 and K2HPO4 (Pi) as substrates. Since the maltotetraose is not active as a substrate for production of G-1-P, effective glucan concentration was defined on a basis of moles of glucose (CeG) excluding a segment of maltotetraose from dextrin. Furthermore, since the reaction was a complicated reversible reaction involving many kinds of glucan differing in chain length, the inhibitory effect of a high concentration of substrate for the enzyme, an apparent equilibrium constant Kapp was defined. Kapp was expressed as a function of initial concentration of glucan (CeG,0) and phosphate (CP,0). The concentration of G-1-P finally obtained at the equilibrium (CG1P,eq) could be easily calculated from Kapp regardless of chain length and branch structures of glucan. As a result, a diagram expressing CG1P,eq as a function of CeG,0 and CP,0 was proposed. A high concentration of G-1-P, namely 230 mM G-1-P was experimentally obtained when CeG,0 and CP,0 were 516 and 3000 mM, respectively, and the enzymatic activity was 100 U·mL–1.

UDP-α- d -glucose pyrophosphorylase (α- d -glucose-1-phosphate uridyltransferase) (UGPase) catalyzes the formation of activated UDP-glucose from glucose-1-phosphate and UTP. Herein we report the cloning and characterization of a thermostable UGPase from a mesophilic, Gram-negative pathogen Helicobacter pylori . An electrospray ionization mass spectrometry (ESI-MS)-based assay demonstrates that the recombinant enzyme absolutely requires divalent cation for its activity, reaching a maximum in the presence of 3 mM Mg 2+ . The optimum activity of the enzyme was around pH 8–9.5 in Tris–HCl buffer and at 37–70 °C; maximum activity was observed at pH 8.5 and 60 °C. Apart from its natural substrate, glucose-1-phosphate, the enzyme also accepts mannose-, galactose-, and glucosamine-1-phosphates. The apparent Michaelis constants of the purified enzyme for glucose-1-phosphate with UTP and dTTP and for mannose-1-phosphate with UTP are 15 ± 2, 32 ± 4 and 77 ± 9 μM, respectively, with the corresponding turnover numbers 5.4, 5.4 and 1.2 min −1 , respectively. An initial velocity study of the forward reaction of the enzyme indicates an ordered bi–bi catalytic mechanism. Analysis of the genomes of other organisms that grow at 37 °C predicts many more such thermostable biocatalysts.

Active calcium transport in intestine is essential for serum calcium homeostasis as well as for bone formation. It is well recognized that vitamin D is a major, if not sole, stimulator of intestinal calcium transport activity in mammals. Besides vitamin D, endogenous glucose 1-phosphate (G1P) affects calcium transport activity in some microorganisms. In this study, we investigated whether G1P affects intestinal calcium transport activity in mammals as well. Of several glycolytic intermediates, G1P was the sole sugar compound in stimulating intestinal calcium uptake in Caco-2 cells. G1P stimulated net calcium influx and expression of calbindin D9K protein in rat intestine, through an active transport mechanism. Calcium uptake in G1P-supplemented rats was greater than that in the control rats fed a diet containing adequate vitamin D3. Bone mineral density (BMD) of aged rat femoral metaphysis and diaphysis was also increased by feeding the G1P diet. G1P did not affect serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] at all. These results suggest that exogenously applied G1P stimulates active transport of calcium in intestine, independent of vitamin D, leading to an increase of BMD.

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.

Increasing antibiotic production yields by favoring the biosynthesis of precursor metabolites glucose-1-phosphate and/or malonyl-CoA in Streptomyces producer strains

Background Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. Results In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. Conclusions Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. General significance This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen., Highlights • Nucleotide-glucose synthesis in Mycobacterium tuberculosis was analyzed. • The characterization of four enzymes involved in glucose-1P partitioning is reported. • Mycobacterial ADP-glucose pyrophosphorylase is allosterically regulated. • Trehalose-6P synthase exhibits higher catalytic efficiency for ADP-glucose. • Trehalose-6P synthase is activated by fructose-6P.

The glgP gene encoding a-glucan phosphorylase (a-GP) from the thermopile Thermus caldophilus GK24 has been identified, cloned, and overexpressed in Escherichia coli and used to synthesize D-glucose-1-phospate (G1P) from an inexpensive starch. The enzyme, purified 6.5-fold, was isolated in 31% yield from the transformed E. coli, and gave a single band. The purified enzyme may exist as a homohexamer with an apparent molecular mass of a 550 kDa molecule, consisting of 90 kDa per subunit. The optimal pH and temperature were 7.0 and 70 8 Ci n thea-GP reaction with starch producing G1P. Soluble starch (amylopectin, amylose) turned out to be a better substrate giving a higher yield of G1P than a-1,6-branched a-1,4-glucans (glycogen, potato starch, etc.). As a result, G1P was obtained in a good yield (47%, w/w) from the reaction containing 5% (w/v) soluble starch in 0.7 M potassium phosphate at pH 7.0. T. caldophilus a-GP shows a high tolerance (up to 0.7 M) of potassium phosphate and plays a critical role in shifting the reaction equilibrium in favor of G1P synthesis. The G1P product can be purified simply by ethanol precipitation, after removing the unreacted starch and inorganic phosphate by activated charcoal and magnesium acetate precipitation. It is concluded that T. caldophilus a-GP readily utilized in large scale synthesis of G1P. # 2005 Elsevier Ltd. All rights reserved.

Aminoglycoside antibiotics are composed of aminosugars and a unique aminocyclitol aglycon including 2-deoxystreptamine (DOS), streptidine, actinamine, etc., and nucleotidylyltransferases, sugar modifying enzymes, and glycosyltransferases appear to be essential for their biosynthesis. However, the genes encoding those enzymes were unable to be identified by a standard homology search in the butirosin biosynthetic btr gene cluster, except that the btrM gene appeared to be a glycosyltransfease. Disruption studies of the btrD gene indicated that BtrD was involved in the supply of a glycosyl donor immediately prior to the glycosylation of DOS giving paromamine. As anticipated, BtrD expressed in Escherichia coli was able to catalyze UDP-D-glucosamine formation from D-glucosamine-1-phosphate and UTP. Both dTTP and UTP were good NTP substrates, and D-glucose-1-phosphate and D-glucosamine-1-phosphate were good sugar phosphates for the enzyme reaction. This finding is the first to identify an enzyme which activates a sugar donor in the DOS-containing antibiotics. Interestingly, BtrD homologues have been reported as functionally unknown open reading frames (ORFs) in the biosynthetic gene clusters for several antibiotics including teicoplanin, balhimycin, chloroeremomycin, and mitomycin C. It appears therefore that gene clusters for antibiotic biosynthesis provide their own nucleotidylyltransferases, and the BtrD homologues are among the secondary metabolism specific enzymes.

The higher plant ADP-glucose pyrophosphorylase (AGPase) is a heterotetramer consisting of two regulatory large subunits (LSs) and two catalytic small subunits (SSs). To further characterize the roles of these subunits in determining enzyme function, different combinations of wildtype LS (LWT) and variant forms (LUpReg1, LM345) were co-expressed with wildtype SS (SWT) and variant forms (STG-15 and Sdevo330) and their enzyme properties compared to those measured for the heterotetrameric wildtype enzyme and SS homotetrameric enzymes. Analysis of the allosteric regulatory properties of the various enzymes indicates that although the LS is required for optimal activation by 3-phosphoglyceric acid and resistance to Pi, the overall allosteric regulatory and kinetic properties are specified by both subunits. Our results show that the regulatory and kinetic properties of AGPase are not simply due to the LS modulating the properties of the SS but, instead, are a product of synergistic interaction between the two subunits.

Phosphorus is an important component of phospholipids, ATP and other compounds. It has been suggested that a higher intake of phosphorus possibly inhibits calcium absorption in the gastrointestinal tract, while casein phosphopeptide and some sugar phosphates have a facilitating effect on calcium absorption. The effect of phosphorus may therefore differ by its form, organic or inorganic. We measured the contents of sugar phosphates in major foods and estimated their dietary intake.Large amounts of glucose 1 phosphate, glucose 6 phosphate, and fructose 6 phosphate in the main glycolytic pathway and a small amount of galactose 1 phosphate were detected.Gender, age, and region are the main factors for differing food intake, so we calculated the daily intake of sugar phosphates as 135mg a day based on data from the Ministry of Health, Labour and Welfare of Japan.

The synthesis of a bicyclo[4.1.0]heptyl analogue of glucose-1-phophate, (1R,2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)-bicyclo[4.1.0]heptan-2-yl dihydrogen phosphate (5) is reported. The synthetic route chosen started with methyl α-D-glucopyranoside and was accomplished in 11 steps with an overall yield of 3%. Compound 5 was tested as a potential substrate of UTP:α-D-glucose-1-phosphate uridylyltransferase, the enzyme that converts glucose-1-phosphate into UDP-glucose. However, the conformationally restricted glucose-1-phosphate analogue 5 was found to be a weakly binding inhibitor, rather than a substrate, of the yeast transferase (12% inhibition at a concentration of 0.1 mmol L–1).Key words: glucose 1-phosphate, inhibition, UTP:α-D-glucose-1-phosphate uridylyltransferase.

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.

A method for the direct determination of kinetic constants for phosphoglucomutase and its phosphorylated products is described. Fourier transform ion cyclotron resonance gas-phase ion/molecule reactions between trimethyl borate and glucose phosphate, phosphorylated at either the 1 or the 6 position, generate mass spectra distinguishable with regard to product ion distribution. A multicomponent quantification method is utilized to determine the composition of a binary mixture of the two positional isomers. Using this method, the conversion between glucose-1-phosphate and glucose-6-phosphate can be directly monitored without the use of coupling enzymes. The values of K m for glucose-1-phosphate and glucose-6-phosphate were determined using the substrate–velocity plot and the Haldane relationship, respectively. Values of V max for both the forward and the reverse directions were measured, and the equilibrium constant for the reversible reaction was determined using this methodology. Kinetic parameters measured correlate well with those obtained using traditional methods. The assay was demonstrated to be accurate and particularly convenient to determine kinetic constants for enzymatic systems that involve the interconversion of phosphorylated positional isomers.

Here, we describe results observed on the simultaneous down-regulation of both isoforms of phosphoglucomutase (EC 5.4.2.2; PGM) within potato (Solanum tuberosum L.) plants. Transgenic plants were created that were inhibited in both activities using the antisense approach. Surprisingly, the metabolic phenotype displayed by these transgenics neither resembles either of the phenotypes observed when only one of the isoforms is inhibited nor displays additive effects of these but is rather more reminiscent of the wild type phenotype. The double transgenic lines display a partial or total recovery in both starch accumulation and starch biosynthetic flux and exhibit wild type levels of intermediary metabolites. Given the dramatic reduction of PGM activity in these lines, and the lack of pleiotropic effects on other enzymes that mediate the sucrose to starch transition, these data strongly suggest a conditional bypass of the normal starch biosynthetic pathway. Based on these data, we present and discuss three models that can be postulated in which the reactions catalysed by the isoforms of PGM can be bypassed. Evaluation of the possible candidates that could mediate this pathway suggests that the uptake of glucose-1-phosphate is the most likely mechanism of alleviating the inhibition of starch synthesis imposed by independent inhibition of either isoform.

The enzyme phosphoglucomutase plays a key role in cellular metabolism by virtue of its ability to interconvert Glc-1-P and Glc-6-P. It was recently shown that a yeast strain lacking the major isoform of phosphoglucomutase (pgm2Delta) accumulates a high level of Glc-1-P and exhibits several phenotypes related to altered Ca(2+) homeostasis when d-galactose is utilized as the carbon source (Fu, L., Miseta, A., Hunton, D., Marchase, R. B., and Bedwell, D. M. (2000) J. Biol. Chem. 275, 5431-5440). These phenotypes include increased Ca(2+) uptake and accumulation and sensitivity to high environmental Ca(2+) levels. In the present study, we overproduced the enzyme UDP-Glc pyrophosphorylase to test whether the overproduction of a downstream metabolite produced from Glc-1-P can also mediate changes in Ca(2+) homeostasis. We found that overproduction of UDP-Glc did not cause any alterations in Ca(2+) uptake or accumulation. We also examined whether Glc-6-P can influence cellular Ca(2+) homeostasis. A yeast strain lacking the beta-subunit of phosphofructokinase (pfk2Delta) accumulates a high level of Glc-6-P (Huang, D., Wilson, W. A., and Roach, P. J. (1997) J. Biol. Chem. 272, 22495-22501). We found that this increase in Glc-6-P led to a 1.5-2-fold increase in total cellular Ca(2+). We also found that the pgm2Delta/pfk2Delta strain, which accumulated high levels of both Glc-6-P and Glc-1-P, no longer exhibited the Ca(2+)-related phenotypes associated with high Glc-1-P levels in the pgm2Delta mutant. These results provide strong evidence that cellular Ca(2+) homeostasis is coupled to the relative levels of Glc-6-P and Glc-1-P in yeast.

糖質を加リン酸分解する酵素はその反応の可逆性を利用して基礎、応用両面から興味が持たれる酵素であるが、加水分解酵素、合成酵素に比べてその研究例は少ない。しかしながら最近になり加リン酸分解酵素の高い立体特異性を生かした糖鎖合成への応用などから新規な加リン酸分解酵素の発見が報告されるようになった。本稿では、これら加リン酸分解酵素の研究例を示すことにより、それらの特性および応用の可能性について概説する。

Cps2L, a thymidylytransferase, is the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis and an antibacterial target. We herein report the evaluation of six sugar phosphate analogues selected to further probe Cps2L substrate tolerance. A modified continuous spectrophotometric assay was employed for facile detection of pyrophosphate (PPi) released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides. Additionally, experiments using waterLOGSY NMR spectroscopy were investigated as a complimentary method to evaluate binding affinity to Cps2L.

The biological and chemical basis of vanadium action in fungi is relatively poorly understood. In the present study, we investigate the influence of vanadate (V5+) on phosphate metabolism of Phycomyces blakesleeanus. Addition of V5+ caused increase of sugar phosphates signal intensities in 31P NMR spectra in vivo. HPLC analysis of mycelial phosphate extracts demonstrated increased concentrations of glucose 6 phosphate, fructose 6 phosphate, fructose 1, 6 phosphate and glucose 1 phosphate after V5+ treatment. Influence of V5+ on the levels of fructose 2, 6 phosphate, glucosamine 6 phosphate and glucose 1, 6 phosphate (HPLC), and polyphosphates, UDPG and ATP (31P NMR) was also established. Increase of sugar phosphates content was not observed after addition of vanadyl (V4+), indicating that only vanadate influences its metabolism. Obtained results from in vivo experiments indicate catalytic/inhibitory vanadate action on enzymes involved in reactions of glycolysis and glycogenesis i.e., phosphoglucomutase, phosphofructokinase and glycogen phosphorylase in filamentous fungi.

Glucose-1-phosphate uridylyltransferase fromErwinia amylovoraCFPB1430 was expressed as a His-tag fusion protein inEscherichia coli. After tag removal, the purified protein was crystallized from 100 mMTris pH 8.5, 2 Mammonium sulfate, 5% ethylene glycol. Diffraction data sets were collected to a maximum resolution of 2.46 Å using synchrotron radiation. The crystals belonged to the hexagonal space groupP62, with unit-cell parametersa= 80.67,b= 80.67,c = 169.18. The structure was solved by molecular replacement using the structure of theE. colienzyme as a search model.

With glucose as the starting material, sodium tripolyphosphate as the phosphorus acylating agent, and urea as the catalyst, glucose 1-phosphate was synthesized and its properties were analyzed. The synthesis conditions were selected according to the phosphorus content and optimized through the response surface methodology (RSM), based on a 3-level, 3-variable Box--Behnken experimental design. The results showed that the phosphorus content of glucose 1-phosphate was 8.12% using a reaction temperature of 70 °C, a molar ratio of glucose to sodium tripolyphosphate of 1.54:1, and catalyst amount of 3.7 g. It coincided well with the experimental value (8.34%). The structure of the product was confirmed by IR, UV, and 1H NMR spectra; electrical conductivity; TG; and DTA. The proposed method has high phosphorus content and low toxicity.