Pyrophosphorylases in Solanum tuberosum (IV. Purification,Tissue Localization,and Physicochemical Properties of UDP-Glucose Pyrophosphorylase)

The enzyme UDP-glucose pyrophosphorylase (UGPase) from potato (Solanum tuberosum L. cv Norchip) tubers was purified 177-fold to near homogeneity and to a specific activity of 1099 international units/mg of protein. The molecular mass of the purified enzyme was 53 kD as determined by SDS-PAGE and gel filtration. Immunological and activity assays detected UGPase at similar levels in potato stems, stolons, and tubers. Leaves and roots contained lower levels of UGPase activity and protein. Lineweaver-Burk plots for substrates inorganic pyrophosphate and UDP-glucose were linear in the pyrophosphorolytic direction, yielding Km values of 0.13 and 0.14 mM, respectively. However, Lineweaver-Burk plots for the substrates glucose-1-P and UTP were biphasic in nature when UGPase was assayed in the direction of UDP-glucose synthesis. At physiological substrate concentrations (i.e. from 0.05-0.20 mM), Km values of 0.08 mM (glucose-1-P) and 0.12mM (UTP) were obtained. When substrate concentrations increased above 0.20 mM, Km values increased to 0.68 mM (glucose-1-P) and 0.53 mM (UTP). These kinetic patterns of potato UGPase suggest a "negative cooperative effect" (A. Conway, D.E. Koshland, Jr. [1968] Biochemistry 7: 4011-4022) with respect to the substrates glucose-1-P and UTP. The biphasic substrate saturation curves were similar to the kinetics of the dimeric form of UGPase purified from Salmonella typhimurium (T. Nakae [1971] J Biol Chem 246: 4404-4411). The in vivo significance of the enzyme's "negative cooperativity" in the direction of UDP-glucose synthesis and potato sweetening is discussed.     

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.     

An improved radiochemical assay for uridine diphosphoglucose pyrophosphorylase

UDP glucose is an important intermediate in numerous metabolic pathways (1). It is therefore not surprising that the enzyme which catalyses its formation, UDP-glucose pyrophosphorylase is ubiquitous (see (2) for references). The reaction catalysed by UDP-glucose pyrophosphorylase is:

$\text{glucose-1}\text{-P +}\text{UTP ? UDP glucose + PPi}$

and the enzyme has been assayed either in the direction of pyrophosphorolysis of the nucleoside diphosphate sugar or in the direction of UDP-glucose formation.Spectrophotometric assays of UDP-glucose pyrophosphorylase in the direction of pyrophosphorolysis are often nonspecific by virtue of the nature of the coupling enzymes (3), whereas similar assays in the direction of UDPG formation may lack the expected stoichiometry of reaction (3,4). Radioisotopic techniques for the assay of UDP-glucose pyrophosphorylase (5,6) are to be preferred to spectrophotometric assays both for their increased sensitivity and specificity. However, these methods depend upon the specific isolation of the radioactive UDP glucose formed, either by a somewhat tedious adsorption to and elution from charcoal (5) or a hazardous precipitation using mercuric acetate. For routine assay of a large number of samples it would be advantageous to replace these techniques with one involving a safer, more rapid method of radioactive UDP-glucose isolation. The radiochemical assay described in this note utilises the binding of UDP glucose to commercially available, anion-exchange filter-paper discs for this purpose. Although the technique was designed to assay UDP-glucose pyrophosphorylase in cell extracts of the cellular slime mould, Dictyostelium discoideum, it should be applicable to most sources of the enzyme.     

The GalF protein of Escherichia coli is not a UDP-glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP-glucose

We report the functional characterization of the galF gene of strain VW187 ( Escherichia coli O7:K1), which encodes a polypeptide displaying structural features common to bacterial UDP-glucose pyrophosphorylases, including the E. coli GalU protein. These enzymes catalyse a reversible reaction converting UTP and glucose-1-phosphate into UDP-glucose and PP_i. We show that, although the GalF protein is expressed in vivo , GalF-expressing plasmids cannot complement the phenotype of a galU mutant and extracts from this mutant which only produces GalF are enzymatically inactive. In contrast, the presence of GalU and GalF proteins in the same cell-free extract caused a significant reduction in the rate of pyrophosphorolysis (conversion of UDP-glucose into glucose-1-phosphate) but no significant effect on the kinetics of synthesis of UDP-glucose. The presence of GalF also increased the thermal stability of the enzyme in vitro. The effect of GalF in the biochemical properties of the UDP-glucose pyrophosphorylase required the co-synthesis of GalF and GalU, suggesting that they could interact as components of the oligomeric enzyme. The physical interaction of GalU and GalF was demonstrated in vivo by the co-expression of both proteins as fusion products using a yeast two-hybrid system. Furthermore, using a pair of galF  ⁺/ galU ⁺ and galF/galU  ⁺ isogenic strains, we demonstrated that the presence of GalF is associated with an increased concentration of intracellular UDP-glucose as well as with an enhancement of the thermal stability of the UDP-glucose pyrophosphorylase in vivo . We propose that GalF is a non-catalytic subunit of the UDP-glucose pyrophosphorylase modulating the enzyme activity to increase the formation of UDP-glucose, and this function is important for bacterial adaptation to conditions of stress.     

The Purification and Some Properties of the UDP-Glucose Pyrophosphorylase from Pollen of Typha latifolia Linne

UDP-glucose pyrophosphorylase from the pollen of Typha latifoliaLinné was purified to homogeneity as judged by polyacrylamidegel electrophoresis. Two affinity chromatographies with UDP-glucoseSepharose 6B and glucose-1-phosphate (G-l-P) Sepharose 6B wereused in the purification. The molecular weight of this enzyme was estimated to be about53,000 by gel filtration and 55,000 by SDS-polyacrylamide gelelectrophoresis, evidence that it consists of a single protein.It had a specific activity of 3,400, a molecular activity of180,000 and an equilibrium constant of 2.1 in the directionof UDP-glucose pyrophosphorolysis. Results of experiments on product inhibition and the initialvelocity of the reaction of the enzyme suggested an orderedBi Bi mechanism. (Received July 8, 1982; Accepted November 12, 1982)     

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.     

Cloning and expression of glucose-1-phosphate thymidylyltransferase gene (schS6) from Streptomyces sp. SCC-2136

The deoxysugar biosynthetic gene cluster of Sch 47554/Sch 47555 was cloned from Streptomyces sp. SCC-2136. One of the ORFs, schS6, appeared to encode glucose-1-phosphate thymidylyltransferase, which converts dTTP and glucose-1-phosphate to TDP-D-glucose and pyrophosphate. The dTDP-D-glucose is a key metabolite in prokaryotics as a precursor for a large number of modified deoxysugars, and these deoxysugars are a major part of various antibiotics, ranging from glycosides to macrolides. SchS6 was expressed in E. coli vector pSCHS6 and the expressed protein was purified to apparent homogeneity by ammonium sulfate precipitation and Ni-NTA affinity column chromatography. The specific activity of the purified enzyme increased 4.7-fold with 17.5% recovery. It migrated as a single band on SDS-PAGE with an apparent molecular mass of 56 kDa. The purified protein showed glucose-1-phosphate thymidylyltransferase activity, catalyzing a reversible bimolecular group transfer reaction. In the forward reaction, the highest activity was obtained with combination of dTTP and alpha-D-glucose-1-phosphate, and only 12% of that activity was obtained with the substrates UTP/alpha-D-glucose-1-phosphate. In the opposite direction, the purified protein was highly specific for dTDP-D-glucose and pyrophosphate.     

UDP-glucose Pyrophosphorylase from Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

UDP-glucose pyrophosphorylase of Jerusalem artichoke tubers was purified 90-fold over the crude extract. The purified enzyme preparation absolutely required magnesium ions for activity. Cobalt ions were 60% as effective as magnesium ions; other divalent cations including manganese showed little or no effect. This enzyme had a pH optimum of 8.5 and a temperature optimum of 40°C. ATP and UDP inhibited the activity of this enzyme in both forward and backward directions. Km values for UDP-glucose, inorganic pyrophosphate, glucose-1-phosphate and UTP were determined to be 4.45 × 10^?4 M, 2.33 × 10^?4 M, 9.38 × 10^?4 M and 2.98 × 10^?4 M, respectively. These results are discussed in comparison with those of UDP-glucose pyrophosphorylases isolated from other plants.     

Preparation of UDP-galacturonic acid using UDP-sugar pyrophosphorylase

UDP-galacturonic acid, the activated form of galacturonic acid (GalUA), is synthesized both de novo and by salvage pathways. The UDP-GalUA pyrophosphorylase gene involved in the salvage pathway has not been identified. Here we show that UDP-sugar pyrophosphorylase from Pisum sativum with a broad specificity has UDP-GalUA pyrophosphorylase activity. The enzyme catalyzed the formation of UDP-GalUA and pyrophosphate from GalUA 1-phosphate and UTP with an equilibrium constant value of 0.24. The recombinant UDP-sugar pyrophosphorylase had optimal pH of 6.0, and the apparent K(m) values for GalUA 1-phosphate, UTP, UDP-GalUA, and pyrophosphate were 2.27, 1.15, 0.70, and 1.26 mM, respectively. In the presence of inorganic pyrophosphatase, the recombinant enzyme produced UDP-GalUA in an 84% yield (based on the GalUA 1-phosphate substrate) on a preparative scale. Thus, this UDP-sugar pyrophosphorylase is useful for the highly efficient production of UDP-GalUA for studies on pectin biosynthesis.     

Expression, purification, and characterization of a functionally active Mycobacterium tuberculosis UDP-glucose pyrophosphorylase

Tuberculosis, which is caused by Mycobacterium tuberculosis, remains to be a global health problem. The thick and complex cell envelope has been implicated in many aspects of the pathogenicity of M. tuberculosis. M. tuberculosis UDP-glucose pyrophosphorylase (UGP, coded by galU, Rv0993) is involved in cell envelope precursor synthesis. UGP catalyzes the reversible formation of UDP-glucose and inorganic pyrophosphate from UTP and glucose 1-phosphate (Glc-l-P). Bacterial UGPs are completely unrelated to their eukaryotic counterparts. This enzyme is recognized as a virulence factor in several bacterial species and is conserved among mycobacterial species, which makes it a good target for mycobacterial pathogenicity research. The recombinant M. tuberculosis UGP (rMtUGP) was purified in Escherichia coli and found to be stable and catalytically active. The effects of pH, temperature and Mg2+ on enzyme activity were characterized. In addition, subcellular localization studies revealed that most of M. tuberculosis UGP protein was located in the cell wall. The purification and characterization of M. tuberculosis UGP may help to decipher the pathogenicity of M. tuberculosis.     

Alteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction

Expanding the scope of stereoselectivity is of current interest in enzyme catalysis. In this study, using error-prone polymerase chain reaction (PCR), a thermostable adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase) from Thermus caldophilus GK-24 has been altered to improve its catalytic activity toward enatiomeric substrates including [glucose-1-phosphate (G-1-P) + uridine triphosphate (UTP)] and [N-acetylglucosamine-1-phosphate (GlcNAc) + UTP] to produce uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine, respectively. To elucidate the amino acids responsible for catalytic activity, screening for UDP-glucose pyrophosphorylase (UGPase) and UDP-N-acetylglucosamine pyrophosphorylase (UNGPase) activities was carried out. Among 656 colonies, two colonies showed UGPase activities and three colonies for UNGPase activities. DNA sequence analyses and enzyme assays showed that two mutant clones (H145G) specifically have an UGPase activity, indicating that the changed glycine residue from histidine has the base specificity for UTP. Also, three double mutants (H145G/A325V) showed a UNGPase, and A325 was associated with sugar binding, conferring the specificity for the sugar substrates and V325 of the mutant appears to be indirectly involved in the binding of the N-acetylamine group of N-acetylglucosmine-1-phosphate. The authors Hosung Sohn and Yong-Sam Kim equally contributed to the study.     

Cloning and expression of the glucose-1-phosphate thymidylyltransferase gene (gerD) from Streptomyces sp. GERI-155

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules, that has antimicrobial activity against Gram-positive bacteria. The deoxysugar biosynthetic gene cluster of GERI-155 was cloned from Streptomyces sp., GERI-155. One of the orfs, gerD, appeared to encode glucose-1-phosphate thymidylyltransferase (dTDP-glucose synthase), which converts dTTP and glucose-1-phosphate to dTDP-D-glucose and pyrophosphate. GerD was expressed in E. coli in vector pHJ2 and the expressed protein was purified to apparent homogeneity by ammonium sulfate precipitation and DEAE-Sepharose CL-6B and DEAE-Trisacryl column chromatography. The specific activity of the enzyme increased 16-fold with a recovery of 10%. It migrated as a single band on SDS-PAGE with a molecular mass of 30 kDa. The purified protein had glucose-1-phosphate thymidylyltransferase activity, catalyzing a reversible bimolecular group transfer reaction. In the forward reaction the highest activity was obtained with the combination of dTTP and alpha-D-glucose-1-phosphate, and only 5.5% of that activity was obtained with UTP in place of dTTP. In the opposite direction the purified protein was highly specific for dTDP-D-glucose and pyrophosphate.     

CugP Is a Novel Ubiquitous Non-GalU-Type Bacterial UDP-Glucose Pyrophosphorylase Found in Cyanobacteria

UDP-glucose pyrophosphorylase synthesizes UDP-glucose from UTP and glucose 1-phosphate and exists in almost all species. Most bacteria possess a GalU-type UDP-glucose pyrophosphorylase, whereas many cyanobacteria species do not. In certain cyanobacteria, UDP-glucose is used as a substrate for synthesis of exopolysaccharide cellulose in spite of the absence of GalU-type UDP-glucose pyrophosphorylase. Therefore, there should be an uncharacterized UDP-glucose pyrophosphorylase in cyanobacteria. Here, we show that all cyanobacteria possess a non-GalU-type bacterial UDP-glucose pyrophosphorylase, i.e., CugP, a novel family in the nucleotide triphosphate transferase superfamily. The expressed recombinant Synechocystis sp. strain PCC 6803 CugP had pyrophosphorylase activity that was highly specific for UTP and glucose 1-phosphate. The fact that the CugP gene cannot be deleted completely in Synechocystis sp. PCC 6803 suggests its central role as the substrate supplier for galactolipid synthesis. Galactolipids are major constituents of the photosynthetic thylakoid membrane and important for photosynthetic activity. Based on phylogenetic analysis, this CugP-type UDP-glucose pyrophosphorylase may have recently been horizontally transferred to certain noncyanobacteria.     

Analysis of glucose 1-phosphate at the picomole level with [C]UTP and uridine 5′-diphosphoglucose pyrophosphorylase

A new sensitive method is described for glucose 1-phosphate analysis. The key reaction is the pyrophosphorolysis of UDP-glucose catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase. The reaction product, [¹⁴C]UDP-glucose, is separated from [¹⁴C]UTP by adsorbing [¹⁴C]UTP selectively onto polyethyleneimine cellulose or by separating both labeled compounds on one-dimensional polyethyleneimine thin-layer chromatograms. The sensitivity of the method for glucose 1-phosphate analysis is 5 pmol. The method has been successfully employed to monitor the level of glucose 1-phosphate in early germination of wheat embryos.     

Control of glucuronidation during hypoxia. Limitation by UDP-glucose pyrophosphorylase.

The regulation of glucuronidation during hypoxia was studied in isolated hepatocytes by analysing the dependence of acetaminophen glucuronidation rate on the intracellular concentrations of UTP, glucose 1-phosphate, UDP-glucose and UDP-glucuronic acid. The steady-state concentrations of these metabolites in cells from fed and starved rats were altered by exposure to various hypoxic O2 concentrations and by adding exogenous glucose. Changes in glucuronidation rate under all conditions were explained in terms of the concentrations of the substrates for UDP-glucose pyrophosphorylase, i.e. UTP and glucose 1-phosphate. Steady-state rates for the UDP-glucose pyrophosphorylase reaction, calculated by using published kinetic constants and measured glucose 1-phosphate and UTP concentrations, were in agreement with the measured glucuronidation rates. Thus the UDP-glucose pyrophosphorylase reaction is the key regulatory site for drug glucuronidation during hypoxia. Control at this site indicates that glucuronidation in vivo may be generally depressed in pathological conditions involving hypoxia and energy (calorie) malnutrition.     

The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate

Background

Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes.

Results

Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification.

Conclusions

Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides.

General significance

The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.     

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

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

Uridine diphosphate glucose synthase from calf liver: determinants of enzyme activity in vitro.

The reaction catalyzed by calf liver uridine diphosphate glucose synthase (pyrophosphorylase) (EC 2.7.7.9; UTP + glucose 1-phosphate = UDP-glucose + PPi) is an example of an enzymic reaction in which a nucleoside triphosphate other than ATP is the immediate source of metabolic energy. Kinetic properties of the enzyme, acting in the direction of UCP-glucose formation were investigated in vitro. The reaction was inhibited by UDP-glucose (0.072), Pi (11), UDP (1.6), UDP-xylose (0.87), UDP-glucuronate (1.3), and UDP-galacturonate (0.95). The numbers in parentheses indicate the concentration (mM) required for half-maximal inhibition under the conditions used. Other compounds tested, including ATP, ADP, and AMP, had no effect. Over a range of concentrations of UTP (0.04-0.8 MM) and UDP-glucose (0.05-0.03 mM), the reaction rate was more dependent on the concentration ratio [UDP-glucose]/[UTP] than on the absolute concentration of either compound. Comparison of the kinetic properties in vitro with estimates of metabolite levels in vivo suggests that (1) the enzyme operates in a range far from its maximal rate, and (2) the concentrations of glucose 1-phosphate and Pi and the ratio [UDP-glucose]/[UTP] may be the most important determinants of UDP-glucose synthase activity.     

Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

Amoebiasis is an intestinal infection caused by the human pathogen Entamoeba histolytica and representing the third leading cause of death by parasites in the world. Host-parasite interactions mainly involve anchored glycoconjugates localized in the surface of the parasitic cell. In protozoa, synthesis of structural oligo- and polysaccharides occurs via UDP-glucose, generated in a reaction catalyzed by UDP-glucose pyrophosphorylase. We report the molecular cloning of the gene coding for this enzyme from genomic DNA of E. histolytica and its recombinant expression in Escherichia coli cells. The purified enzyme was kinetically characterized, catalyzing UDP-glucose synthesis and pyrophosphorolysis with V_max values of 95 U/mg and 3 U/mg, respectively, and affinity for substrates comparable to those found for the enzyme from other sources. Enzyme activity was affected by redox modification of thiol groups. Different oxidants, including diamide, hydrogen peroxide and sodium nitroprusside inactivated the enzyme. The process was completely reverted by reducing agents, mainly cysteine, dithiothreitol, and thioredoxin. Characterization of the enzyme mutants C94S, C108S, C191S, C354S, C378S, C108/378S, M106S and M106C supported a molecular mechanism for the redox regulation. Molecular modeling confirmed the role of specific cysteine and methionine residues as targets for redox modification in the entamoebic enzyme. Our results suggest that UDP-glucose pyrophosphorylase is a regulated enzyme in E. histolytica. Interestingly, results strongly agree with the occurrence of a physiological redox mechanism modulating enzyme activity, which would critically affect carbohydrate metabolism in the protozoon.     

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).