UDP-glucose pyrophosphorylase from potato tuber: purification and characterization

UDP-glucose pyrophosphorylase from potato tuber was purified 243-fold to a nearly homogeneous state with a recovery of 30%. The purified enzyme utilized UDP-glucose, but not ADP-glucose, as the substrate, and was not activated by 3-phosphoglyceric acid. Product inhibition studies revealed the sequential binding of UDP-glucose and MgPPi and the sequential release of glucose-1-phosphate and MgUTP, in this order. Analyses of the effects of Mg2+ on the enzyme activity suggest that the MgPPi and MgUTP complexes are the actual substrates for the enzyme reaction, and that free UTP acts as an inhibitor. The enzyme exists probably as the monomer of an approximately 50-kDa polypeptide with a blocked amino terminus. For structural comparison, 29 peptides isolated from a tryptic digest of the S-carboxymethylated enzyme were sequenced. The results show that the potato tuber enzyme is homologous to UDP-glucose pyrophosphorylase from slime mold, but not to ADP-glucose pyrophosphorylase from Escherichia coli, and provide structural evidence that UDP-glucose and ADP-glucose pyrophosphorylase are two different protein entities.     

Soluble adenosine diphosphate glucose–α-1,4-glucan α-4-glucosyltransferases from spinach leaves

Multiple forms of ADP-glucose-alpha-1,4-glucan alpha-4-glucosyltransferase were obtained from spinach leaves by gradient elution from a DEAE-cellulose column. In the presence of high concentrations of some salts and bovine serum albumin, unprimed activity was found in one (transglucosylase III) of the four fractions eluted from the column. In addition to having unprimed activity, transglucosylase III had a lower K(m) for ADP-glucose, a much higher K(m) for oyster glycogen, greater heat sensitivity and lower affinity for maltose, maltotriose and amylopectin beta-limit dextrin than fractions I, II and IV. In addition, the kinetics at low concentrations of amylose, amylopectin and rabbit liver glycogen were non-linear for transglucosylase III. The properties of transglucosylases I, II and IV were generally similar to each other. Rates of the unprimed reaction at physiological concentrations of ADP-glucose were greater than those found for the primed reaction of fraction III. The product formed by the unprimed reaction was a glucan containing principally alpha-1,4 linkages with some alpha-1,6 linkages. The primer, maltose, at a concentration of 0.5m inhibited the synthesis of the unprimed product.     

Sucrose Synthetase of Sweet Potato Roots

The effect of concentration of each substrate in the reaction catalyzed by sucrose synthetase isolated from sweet potato roots was determined. For the sucrose synthesizing reaction, UDP-glucose(ADP-glucose)+fructose→sucrose+UDP(ADP), the substrate saturation curves for UDP-glucose, ADP-glucose and fructose were hyperbolic in shape and the reaction was strongly inhibited by UDP competitively. On the other hand, the substrates for the reversal of sucrose synthetase reaction, sucrose+UDP(ADP)→UDP-glucose(ADP-glucose)+fructose, exhibited a sigmoidal shaped saturation curve which was deviated from the Michaelis-Menten equation. The plot of data according to the empirical Hill equation gives a values greater than 1.0 for every substrate examined in the latter case. In view of these experimental data, the major role of sucrose synthetase is postulated in that this enzyme is involved in the breakdown of sucrose in sweet potato root tissues instead of the sucrose synthesizing reaction. The molecular weight of the enzyme was determined to be about 540,000 by the Sephadex gel filtration chromatography.     

Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane

In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.     

Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme.

The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli ( approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.     

Amylopectin Synthase of Eimeria tenella: Identification and Kinetic Characterization

ABSTRACT. A soluble enzyme amylopectin synthase (UDP-glucose-α 1,4-glucan α-4-glucosyltransferase) which transfers glucose from uridine 5'-diphosphate glucose (UDP-glucose) to a primer to form α-I,4-glucosyl linkages has been identified in the extracts of unsporulated oocysts of Eimeria tenella . UDP-glucose and not ADP-glucose was the most active glucosyl donor. Corn amylopectin, rabbit liver glycogen, oyster glycogen and corn starch served as primers; the latter two were less efficient. The enzyme has an apparent pH optimum of 7.5 and exhibited typical Michaelis-Menten kinetics with dependence on both the primer and substrate concentrations. The Michaelis constants (Km). with respect to UDP-glucose, was 0.5 mM; and 0.25 mg/ml and 1.25 mg/ml with respect to amylopectin and rabbit liver glycogen. The product formed by the reaction was predominantly a glucan containing α-1,4 linkages. The specificity of the enzyme suggests that this enzyme is similar to glycogen synthase in eukaryotes and has been designated as amylopectin synthase (UDP-glucose-α-1,4-glucosetransferase EC 2.4.1.11).     

Some Properties of Potato Tuber UDPGd-fructose-2-glucosyltransferase (E.C. 2.4.1.14) and UDPGd-fructose-6-phosphate-2-glucosyltransferase (E.C. 2.4.1.13)

Sucrose and sucrose 6-phosphate synthetase were isolated from potato tubers, partially purified and their properties studied. The sucrose synthetase showed optimum activity at 45° and was inhibited competitively by ADP and some phenolic glucosides. The Ki′s for these inhibitors were determined. Mg²⁺ was found to activate this enzyme. Activity toward UDP-glucose or ADP-glucose formation was measured. The optimum conditions for sucrose and UDP-glucose formation were found to differ. The specificity for the glucosyl donor and acceptor were determined.

The optimum conditions for sucrose 6-phosphate synthetase activity were studied. This enzyme was not inhibited by either ADP or phenolic glucosides; UDP-glucose was the only glucosyl donor for sucrose 6-phosphate formation.

     

Pyrophosphorylases in Solanum tuberosum: I. Changes in ADP-Glucose and UDP-Glucose Pyrophosphorylase Activities Associated with Starch Biosynthesis during Tuberization, Maturation, and Storage of Potatoes

Changes in ADP-glucose and UDP-glucose pyrophosphorylase activities were followed during tuber development of Solanum tuberosum and prolonged storage at 4 and 11 C. Potato tuberization was accompanied by a sharp increase in starch synthesis simultaneous with a marked rise in ADP-glucose pyrophosphorylase activity. When tubers reached an average diameter of 1 centimeter (0.5 gram average tuber weight) and had already established 58% starch on a dry weight basis, ADP-glucose pyrophosphorylase increased 16- to 24-fold over its activity seen in low starch containing stolon tissue. During this same period UDP-glucose pyrophosphorylase increased approximately 2- to 3-fold. Although participation of UDP-glucose in starch formation can not be neglected, it is suggested that the onset of rapid non-photosynthetic potato tuber starch biosynthesis may be closely related to the simultaneous increase in ADP-glucose pyrophosphorylase activity.     

Partial Purification and Some Properties of ADP-glucose Phosphorylase from Potato Tubers

ADP-glucose phosphorylase [adenosine diphosphate glucose: orthophosphate adenyl- yltransferase; Dankert et ah, Biochim. Biophys. Acta, 81, 78 (1964)] was found to be widely distributed in plant tissues. The enzyme was purified 570-fold in a 24% yield from cell- free extract of growing tubers of potato (Solanum tuberosum L.). The following reaction catalyzed by the purified enzyme was found to proceed stoichiometrically. ADP-glucose +P₁→ADP+glucose-1-P

Maximal activity was observed at pH 8. The enzyme was the most stable at pH 7, showing 50% loss of its original activity after 50 min heating at 57°C. The following kinetic parameters were obtained: activation energy, 11.1 kcal/mole; Km (P₁), 2.5 mm; Km (ADP-glucose), 0.05 mm. The enzyme did not act on GDP-mannose, GDP-glucose and UDP-glucose. Neither activator nor inhibitor was found among various phosphorylated metabolites tested. The enzyme was inhibited by metal-binding reagents, EDTA and o-phenanthroline. None of the metal ions tested was found to recover the activity of chelator-treated enzyme.     

Potato Tuber UDP-Glucose:Protein Transglucosylase Catalyzes Its Own Glucosylation

Potato (Solanum tuberosum L.) tuber UDP-glucose:protein transglucosylase (UPTG) (EC 2.4.1.112) is involved in the first of a two-step mechanism proposed for protein-bound α-glucan synthesis by catalyzing the covalent attachment of a single glucose residue to an acceptor protein. The resulting glucosylated 38-kilodalton polypeptide would then serve as a primer for enzymic glucan chain elongation during the second step. In the present report, we describe the fast protein liquid chromatography purification of UPTG from a membrane pellet of potato tuber. An apparently close association of UPTG, phosphorylase, and starch synthase was observed under native conditions during different purification steps. Enrichment of a 38-kilodalton polypeptide was found throughout enzyme purification. It is now shown that the purified UPTG, with an apparent molecular mass of 38 kilodaltons, undergoes self-glucosylation in a UDP-glucose- and Mn²⁺-dependent reaction. Therefore, it is concluded that UPTG is the enzyme and at the same time the priming protein required for the biogenesis of protein-bound α-glucan in potato tuber.     

UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing

We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemically [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.     

Enzymatic Glucosylation of Dolichyl Monophosphate Formed Via Cytidine Triphosphate in Calf Brain Membranes

When calf brain membrane preparations containing endogenous dolichyl [32P]monophosphate (Dol-32P), prelabeled enzymatically by [gamma-32P]-CTP, are incubated with unlabeled UDP-glucose, the formation of a mild acid-labile [32P]phosphoglucolipid is observed. The biosynthesis of the [32P]phosphoglucolipid is dependent on the concentration of UDP-glucose added, and no [32P]phosphoglycolipid appeared when UDP-glucose was replaced by ADP-glucose, UDP-xylose, UDP-galactose, UDP-mannose, or UDP-glucuronic acid. The 32P-labeled product formed by the UDP-glucose-dependent reaction is chemically and chromatographically identical to glucosylphosphoryldolichol. Several enzymatic parameters of the glucosylation of the specific pool of Dol-P, synthesized by the CTP-mediated kinase, and the total available pool of Dol-P have been compared by a double-label assay utilizing endogenous, prelabeled Dol-32P and UDP-[3H]glucose as substrates.     

A new ADP (UDP)--glucose glucosyltransferase activity in disrupted starch grains.

Disrupted potato starch granules obtained in the presence of 8 M urea were shown to increase [¹⁴C] glucose incorporation from labeled ADP-glucose or UDP-glucose into starch, as compared to intact grains. Labeled glucose or maltose units were found to be incorporated through a linkage that produced cyclic phosphate esters upon mild alkaline treatment and was sensitive to hydrolysis at pH 2.0. Both properties of this linkage strongly resembled those of the pyrophosphate bond of ADP-glucose or UDP-glucose.     

Composition of Soluble Nucleotides in Growing Corms of Amorphophallus konjac C. Koch

The nucleotides and the activities of both sucrose synthetase and granular starch synthetase in the konjak corm (Amorphophallus konjac C. Koch) have been investigated as a preliminary experiment on konjak mannan biosynthesis. On chromatographic separation on anion exchange resin and paper of compounds present in the acid ethanol extract from the corms, ascorbic acid, AMP, ADP, ATP, ADP-glucose, UTP, UDP-glucose, GTP, and GDP-mannose were isolated and tentatively identified. An unidentified nucleotide was also isolated.

Of the three nucleotide sugars, UDP-glucose was the most plentiful, while ADP-glucose was the least. The sucrose synthetase in konjak corms was as active as that in other plants. These observations suggest that the mechanism involved in sucrose cleavage in konjak corms is the same as that in other plants, such as sweet potato roots. Starch synthetase bound to starch granules in konjak corms was also found to be active when ADP-glucose was used as glucose donor. But UDP-glucose could not be substituted for ADP-glucose.

Based on these observations the mechanism of konjak mannan biosynthesis is discussed.     

An efficient chemoenzymatic production of small molecule glucosides with in situ UDP-glucose recycling

A one-pot system for efficient enzymatic synthesis of curcumin glucosides is described. The method couples the activities of two recombinant enzymes, UDP-glucose: curcumin glucosyltransferase from Catharanthus roseus (CaUGT2) and sucrose synthase from Arabidopsis thaliana (AtSUS1). UDP, a product inhibitor of UDP-glucosyltransferase, was removed from the system and used for regeneration of UDP-glucose by the second enzyme, AtSUS1. The productivity was increased several-fold and UDP-glucose initially added to the reaction mixture could be reduced to one-tenth of the normal level. The concept of enhancing glucosylation efficiency by coupling a UDP-glucose regeneration system with glucosyltransferases should be applicable to enzymatic production of a wide range of glucosides.     

A Transglucosylase from Sclerotinia libertiana

A Sclerotinia enzyme preparation with predominant exo-beta-(1-->3)-glucanase activity has the capacity to mediate the formation of tetrasaccharide from 3-O-beta-cellobiosyl-d-glucose or cellotriose, and a pentasaccharide from 3-O-beta-cellotriosyl-d-glucose or cellotetraose. Transglucosylation is not observed when the enzyme is incubated in the presence of laminaritriose, laminaritetraose, or cellobiose. Substrate specificity of the reaction therefore resembles certain features of exo-beta-(1-->4)-glucanases. The optimum pH of the activity is 5.5 and the reaction is inhibited by nojirimycin but not by glucono-1,5-lactone. In contrast to the Sclerotinia glucanase, a Basidiomycete exo-beta-(1-->3)-glucanase has no apparent transglucosylase activity. The results indicate that a transglucosylase may have been an undetected constituent in exo-beta-(1-->3)-glucanase preparations used for promoting growth in auxin-depleted tissues.     

Enzymic synthesis of floridean starch in a red alga, Serraticardia maxima

ADP-glucose: -l,4-glucan -4-glucosyltransferase was obtainedfrom a marine red alga Serraticardia maxima in a form boundwith floridean starch granules. The enzyme catalyzed the transferof glucosyl residue from ADP-glucose, UDP-glucose and GDP-glucoseto floridean starch added as a primer. ADP-glucose was the mostefficient glucosyl donor in the reaction. Maltose was producedby ß-amylolysis of the glucan synthesized by the algalenzyme. The optimum pH for enzyme activity was at 8.4. The enzymewas not obtained in a soluble form from either the chloroplastextract or the whole algal cell extract. Electron micrographsof algal cells revealed that floridean starch granules are localizedexclusively outside chloroplasts. Hence, it appears that mostof the synthetase is present outside chloroplasts. ¹ Contribution from the Shimoda Marine Biological Station, TokyoKyoiku University, No. 202. This work was supported by a Grant-in-Aidfor Cooperative Research from the Ministry of Education, Japan. ² Present address: Department of Biology, Faculty of Science,Science University of Tokyo, Kagurazaka, Tokyo 162, Japan. ³ Present address: Laboratory of Biology, Faculty of Science,Toho University, Narashino, Chiba 275, Japan. (Received May 25, 1970; )     

A rapid and simple assay method for UDP-glucose:ceramide glucosyltransferase.

We have developed a simple rapid method for measuring UDP-glucose:ceramide glucosyltransferase; the method utilizes ceramide immobilized on the surface of silica gel and [14C]UDP-glucose as substrate. The reaction product, [14C]glucosylceramide, formed on the surface of the silica gel was easily separated from free [14C]UDP-glucose, either by centrifugation or by filtration. The reliability of this solid phase method was evaluated by using rat brain membrane fraction as an enzyme source. This enzyme had an optimal pH of 6.4-6.5 and required Mn2+, Mg2+ in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Apparent Km values of 8.7 microM for UDP-glucose and 292 microM for ceramide were determined using the new method. Under the optimal conditions, the solid phase method yielded 2-5-times more product than did the method using micellar system. Moreover, the reaction was highly quantitative in its enzyme dose-activity relationship.     

Investigation of the UDP-glucose dehydrogenase reaction for a coupled assay of UDP-glucose pyrophosphorylase activities.

An optimized coupled enzyme assay for UDP-glucose pyrophosphorylase (EC 2.7.7.9) using UDP-glucose dehydrogenase (EC 1.1.1.22) is presented. This optimized assay was developed by a detailed investigation of the kinetics of the UDP-glucose dehydrogenase reaction. In addition the data provide a basis for the enzymatic synthesis of UDP-glucuronic acid. The results demonstrate that the two binding sites of the dehydrogenase differ since a different modulation of the enzyme activity and stability is observed after preincubation with UDP-glucose or NAD+ at various pH values. This is of general interest for the preparation of assay mixtures where UDP-glucose dehydrogenase is used as an auxiliary enzyme.     

Properties of Solubilized UDP-GlcNAc: Dolichyl Phosphate-GlcNAc-1-P-Transferase from Soybean Cultured Cells

The GlcNAc-1-P-transferase was solubilized from microsomal preparations of soybean cultured cells by treatment with 1% Triton X-100. The solubilized enzyme catalyzed the formation of dolichyl pyrophosphoryl-GlcNAc when incubated with UDP-GlcNAc and dolichyl phosphate. The GlcNAc-1-P-transferase activity was stimulated by the addition of phosphatidylglycerol and phosphatidylinositol, but was inhibited by phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The K_m value for dolichyl-phosphate was 6.2 micromolar and that determined for UDP-GlcNAc was 0.42 micromolar. The pH optimum for the GlcNAc-1-P reaction was between 7.2 and 7.6; maximum activity occurred at about 10 millimolar Mg²⁺. The addition of unlabeled GDP-mannose or UDP-glucose considerably inhibited enzyme activity which could be restored to nearly the original value by addition of more dolichyl phosphate to the incubation mixture. On the other hand, the addition of unlabeled ADP-glucose and GDP-glucose enhanced the enzyme activity. This stimulation by these sugar nucleotides was found to be due to the protection of the substrate UDP-[³H]-GlcNAc from pyrophosphatase degradation. The GlcNAc-1-P-transferase reaction was very sensitive to tunicamycin and 50% inhibition required less than 1 microgram of antibiotic per milliliter. Amphomycin, showdomycin, and diumycin also inhibited this reaction but at higher concentrations.