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.     

Dominant ptsH mutations of the gene coding for synthesis of the HPr protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli K-12 merodiploids

Abstract The Escherichia coli ptsI and ptsH genes code for the synthesis of two proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), namely enzyme I and protein HPr. A number of ptsI ⁺ ptsH ⁺/F' ptsI ⁺ ptsH merodiploids was obtained. It was shown in experiments in vivo that ptsH mutations in the transposition are dominant. Bacterial extracts from these merodiploids supported [¹⁴C]methyl glucoside (MG) phosphorylation at the expense of phosphoenolpyruvate only half as much as extracts from the pts ⁺ cells. ptsI ⁺ ptsH /F' ptsI ⁺ ptsH ⁺ merodiploids appeared to be non-viable; the reason for this lack of viability is discussed.     

Genetic transformation using Agrobacterium rhizogenes

UDP-glucose pyrophosphorylase (EC 2.7.7.9) has been highly purified from the plant fraction of soybean ( Glycine max L. Merr. cv Williams) nodules. The purified enzyme gave a single polypeptide band following sodium docecyl sulphate polyacryla-mide gel electrophoresis, but was resolved into three bands of activity in non-denaturing gels. The enzyme appeared to be a monomer of molecular weight between 30 and 40 kDa. UDP-glucose pyrophosphorylase had optimum activity at pH 8.5 and displayed typical hyperbolic kinetics. The enzyme had a requirement for divalent metal ions, and was highly specific for the substrates pyrophosphate and UDP-glucose in the pyrophosphorolysis direction, and glucose-1-phosphate and UTP in the direction of UDP-glucose synthesis. The K_m values were 0.19 m M and 0.07 m M for pyrophosphate and UDP-glucose, respectively, and 0.23 m M and 0.11 m M for glucose-1-phosphate and UTP. The maximum velocity in the pyrophosphorolysis direction was almost double that for the reverse reaction. UDP-glucose pyrophosphorylase did not appear to be subject to a high degree of fine control, and activity in vivo may be regulated mainly by the availability of the substrates.     

Probing the pyrophosphate-binding site in potato tuber UDP-glucose pyrophosphorylase with pyridoxal diphosphate.

Potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose 1-phosphate and MgUTP, according to an ordered bi-bi mechanism in which UDP-glucose and MgPPi bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5'-diphosphate, a reactive PPi analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg2+ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPPi, and glucose 1-phosphate, but enhanced by UDP-glucose. The enhancement was prevented by co-addition of Pi, the competitive inhibitor with respect to PPi. The complete inactivation corresponded to the incorporation of 0.9-1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP-glucose, labels were almost exclusively incorporated into Lys-329. Thus, this residue may be located near the bound MgPPi and its modification is promoted, probably through conformational changes, by the binding of UDP-glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys-329 and Lys-263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPPi and in the UDP-glucose-induced conformational changes, respectively.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

Purification and characterization of an ATP-dependent protein kinase from Streptococcus faecalis

Abstract An enzyme catalyzing the ATP-dependent phosphorylation of HPr of the bacterial phosphotransferase system has been purified from Streptococcus faecalis . Size exclusion chromatography and sodium dodecyl sulfate (SDS) polyacrylamide gels revealed an M _r of 65000. Beside HPr of S. faecalis the protein kinase also phosphorylates HPr of Streptococcus lactis, Streptococcus pyogenes, Bacillus subtilis and Streptococcus aureus , but not HPr of Escherichia coli . The kinase is largely inhibited by P_i and EDTA. Mg²⁺ and Mn²⁺ could overcome inhibition by EDTA. 2-Phosphoglycerate and glucose-6-phosphate, previously reported to stimulate kinase activity in crude extracts, had no effect on the purified enzyme. Fructose-1,6-diphosphate stimulated the protein kinase.     

The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli

In Escherichia coli , lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5 mM EGTA was compensated for by the addition of Zn²⁺. In the presence of 0.5 mM EGTA, only the parental strain was able to take up ⁶⁵Zn²⁺. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI ) and localized at 42 min on the genetic map of E. coli . At high Zn²⁺ concentrations, the znu mutants took up more ⁶⁵Zn²⁺ than the parental strain. The high-affinity ⁶⁵Zn²⁺ uptake was repressed by growth in the presence of 10 μM Zn²⁺. A znuA–lacZ operon fusion was repressed by 5 μM Zn²⁺ and showed a more than 20-fold increase in β-galactosidase activity when Zn²⁺ was bound to 1.5 μM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn²⁺-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli . A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity ⁶⁵Zn²⁺ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB .     

In vitro and in vivo regulation of chloroplast glyceraldehydes-3-phosphate dehydrogenase isozymes from Chenopodium rubrum. I. Purification and properties of isozymes

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13) was purified from leaves of Chenopodium rubrum L. Aggregated (≥ 10⁶) and disaggregated (165 × 10³) molecular weight forms were obtained by gel filtration in the presence of NAD⁺ and NADP⁺, respectively. The disaggregated enzyme was separated into two isozymes by inverse ammonium sulphate gradient solubilization: "NADP-GPD I" was homotetrameric (subunit molecular weight 39 × 10³); "NADP-GPD II" was heterotetrameric (subunit molecular weights 39 × 10³ and 43 × 10³). Isoelectric focusing of the isozymes, both aggregated and disaggregated, revealed two isoelectric forms in each case, at 4.3 and 7.7. Chloroplast GPD was "NADP-suppressed" in crude extracts due to partial oxidation, incubation with dithioerythritol restored full activity.     

Heterologous expression of Escherichia coli ppsA (phosphoenolpyruvate synthetase) and galU (UDP-glucose pyrophosphorylase) genes in Corynebacterium glutamicum, and its impact on trehalose synthesis

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on a Corynebacterium glutamicum strain expressing the Escherichia coli enzyme UDP-glucose pyrophosphorylase (GalU). Biochemical network analysis suggest a further bottleneck for trehalose synthesis resulting from the coupling of phosphotransferase (PTS) mediated glucose uptake, and glucose catabolism in C. glutamicum. To overcome this coupling, we propose the expression of E. coli phosphoenolpyruvate synthetase (PpsA), in addition to GalU expression, in C. glutamicum. Although GalU expression improved trehalose synthesis in C. glutamicum, the simultaneous expression of GalU and PpsA did not result in a further increase in trehalose yield, but resulted in an increased catabolic rate of glucose, which could be ascribed to the operation of a futile cycle between phosphoenolpyruvate and pyruvate. The impact of GalU and PpsA expression on polysaccharide content, side product excretion and metabolic fluxes is discussed, as well as alternative ways to decouple glucose uptake and catabolism, in order to increase trehalose yield.     

Enhanced UDP-glucose and UDP-galactose by homologous overexpression of UDP-glucose pyrophosphorylase in Lactobacillus casei

UDP-sugars are widely used as substrates in the synthesis of oligosaccharides catalyzed by glycosyltransferases. In the present work a metabolic engineering strategy aimed to direct the carbon flux towards UDP-glucose and UDP-galactose biosynthesis was successfully applied in Lactobacillus casei. The galU gene coding for UDP-glucose pyrophosphorylase (GalU) enzyme in L. casei BL23 was cloned under control of the inducible nisA promoter and it was shown to be functional by homologous overexpression. Notably, about an 80-fold increase in GalU activity resulted in approximately a 9-fold increase of UDP-glucose and a 4-fold increase of UDP-galactose. This suggested that the endogenous UDP-galactose 4-epimerase (GalE) activity, which inter-converts both UDP-sugars, is not sufficient to maintain the UDP-glucose/UDP-galactose ratio. The L. casei galE gene coding for GalE was cloned downstream of galU and the resulting plasmid was transformed in L. casei. The new recombinant strain showed about a 4-fold increase of GalE activity, however this increment did not affect that ratio, suggesting that GalE has higher affinity for UDP-galactose than for UDP-glucose. The L. casei strains constructed here that accumulate high intracellular levels of UDP-sugars would be adequate hosts for the production of oligosaccharides.     

l-Myo-inositol-1-phosphate synthase from lower plant groups: Partial purification and properties of the enzyme from Euglena gracilis

A survey for the enzyme L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been conducted among various members of the lower plant groups, mainly algac, bryophytes and fungi; some properties of the partially purified enzyme from Euglena gracilis Z . are presented. The enzyme was detected in Chloropycean algae, Marchantiales and the Basidiomycetous fungi. The enzyme from Euglena had a pH optimum at 7.5. The Km for glucose-6-P was 2.1 m M and for NAD⁺ 80 μ M . When assayed in the absence of added NAD⁺, the enzyme showed a basal activity suggesting the presence of bund NAD⁺ in the system. NH₄Cl increased the enzyme activity two-fold, altough the enzyme was inactivated by (NH₄)₂SO₄.     

Functional analysis of the Lactococcus lactis galU and galE genes and their impact on sugar nucleotide and exopolysaccharide biosynthesis.

We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in L. lactis. The lactococcal galU gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The L. lactis galU gene could complement an Escherichia coli galU mutant, and overexpression of this gene in L. lactis under control of the inducible nisA promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDP-glucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the galE gene resulted in poor growth, undetectable intracellular levels of UDP-galactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the galE disruption mutant, while the level of EPS production was approximately one-half the wild-type level.     

Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of σ32in vivo

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of σ³², an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades σ³² and has a central role in the control of the σ³² level. The ftsH null mutant was isolated, and establishment of the Δ ftsH mutant allowed us to investigate control mechanisms of the stability and the activity of σ³² separately in vivo . Loss of the FtsH function caused marked stabilization and consequent accumulation of σ³² (≈20-fold of the wild type), leading to the impaired downregulation of the level of σ³². Surprisingly, however, Δ ftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of σ³² when it is accumulated. Overproduction of DnaK/J reduces the activity of σ³² in Δ ftsH cells without any detectable changes in the level of σ³², indicating that the DnaK chaperone system is responsible for the activity control of σ³² in vivo . In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of σ³².     

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).     

In vitro and in vivo regulation of chJoroplast glyceraldehyde-3-phosphate dehydrogenase isozymes from Chenopodium rubrum. III. The molecular basis of the aggregation phenomenon: chloroplast glyceraldehyde-3-phosphate dehydrogenase as an ambiquitous enzyme

The nature of the aggregated form of chloroplast glyceraldehyde-3-phosphate dehydrogenase isozymes (GPD, EC 1.2.1.13) from Chenopodium rubrum leaves was investigated. After disaggregation of the isozymes in NADP ⁺ buffer, and resuspension of the disaggregated isozymes in NAD⁺ buffer, complete reaggregation could only be achieved by remixing the enzyme with a high molecular weight fraction, from which the isozymes had dissociated during the NADP⁺ filtration. After separation of the isozymes by inverse ammonium sulphate gradient solubilization, spontaneous extensive reaggregation of each isozyme was observed in NAD⁺ buffer. The high molecular weight material consisted of ribonucleoprotein, and RNase treatment impaired its ability to promote reaggregation of chloroplast GPD. It is proposed that pyridine nucleotide-controlled aggregation and binding to ribonucleoprotein in vitro are artifacts which reflect an in situ binding to cellular components. Since uncontrolled NAD⁺-linked activities of the bifunctional isozymes in the chloroplast would lead to an equalization of the NAD ⁺ and NADP ⁺ redox couples, it is suggested that the reversible binding of the isozymes forms the basis of a regulatory system in vivo.     

UDP-sugar hydrolase isozymes in Salmonella enterica and Escherichia coli: Silent alleles of ushA in related strains of Group I Salmonella isolates, and of ushB in wild-type and K12 strains of E. coli, indicate recent and early silencing events, respectively

Abstract Escherichia coli contains a single periplasmic UDP-glucose hydrolase (5'-nucleotidase) encoded by ushA. Salmonella enterica , serotype Typhimurium, also contains a single UDP-glucose hydrolase but, in contrast to E. coli , it is membrane-bound and is encoded by the non-homologous ushB gene; Salmonella enterica (Typhimurium) also contains a silent allele of the ushA gene ( ushA⁰ ). In this report, we show that nearly all natural isolates of Salmonella contain both UDP-sugar hydrolases, i.e. they are UshA⁺ UshB⁺. The only exceptions are all from sub-group I ( S. gallinarum, S. pullorum , and most Typhimurium strains), are UshA⁻ UshB⁺, and several have been shown to contain an ushA⁰ allele. These data, together with the fact that these latter strains are closely related genetically, strongly suggests a recent silencing mutation(s). We also report the presence in E. coli K-12, and in natural isolates of E. coli , of a DNA sequence which is homologous to the ushB gene of Salmonella ; since E. coli does not contain UshB activity, we tentatively refer to this sequence as ushB⁰ . Since all E. coli strains investigated are UshB⁻, we conclude that the silencing mutation(s) occured relatively eary following the divergence of Escherichia coli and Salmonella from a common ancestor that was ushA⁺ ushB⁺ .     

Strain improvement and metabolic flux modeling of wild-type and mutant <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. NX-3 for synthesis of exopolysaccharide welan gum

Low-energy nitrogen ion beam implantation technique was used for the strain improvement of Alcaligenes sp. NX-3 for the production of exopolysaccharide welan gum. A high welan gum producing mutant, Alcaligenes sp. NX-3-1, was obtained through 20 keV N⁺ ion beam irradiation. Starting at a concentration of 50 g/L of glucose, mutant NX-3-1 produced 25.0 g/L of welan gum after 66 h of cultivation in a 7.5 L bioreactor, which was 34.4% higher than that produced by the wild-type strain. The results of metabolic flux analysis showed that the glucose-6-phosphate and acetyl coenzyme A nodes were the principle and flexible nodes, respectively. At the glucose-6-phosphate node, the fraction of carbon measured from glucose-6-phosphate to glucose-1-phosphate was enhanced after mutagenesis, which indicated that more flux was used to synthesize welan gum in the mutant. By analyzing the activities of related enzymes in the biosynthetic pathway of sugar nucleotides essential for welan gum production, we found that the specific activities of phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-glucose dehydrogenase, and dTDP-glucose pyrophosphorylase in the mutant strain were higher than those in the wild-type strain. These improvements in enzyme activities could be due to the affected of ion beam implantation.     

Sodium Transport from Blood to Brain: Inhibition by Furosemide and Amiloride

Abstract: Brain sodium uptake in vivo was studied using a modified intracarotid bolus injection technique in which the uptake of ²²Na ⁺ was compared with that of the relatively impermeable molecule, [³H]l-glucose. At a Na ⁺ concentration of 1.4 m M , Na ⁺ uptake was 1.74 ± 0.07 times greater than l -glucose uptake. This decreased to 1.34 ± 0.04 at 140 m M Na ⁺, indicating saturable Na ⁺ uptake. Relative Na ⁺ extraction was not affected by pH but was inhibited by amiloride ( K _i= 3 ± 10⁻⁷ M ) and by 1 m M furosemide. The effects of these two inhibitors were additive. Brain uptake of ⁸⁶Rb ⁺, a K ⁺ analogue, was measured to study interaction of K ⁺ with Na ⁺ transport systems. Relative ⁸⁶Rb ⁺ extraction was also inhibited by amiloride; however, it was not inhibited by furosemide. The results suggest the presence of two distinct transport systems that allow Na ⁺ to cross the luminal membrane of the brain capillary endothelial cell. These transport systems could play an important role in the movement of Na ⁺ from blood to brain.     

Induction of autolysis in starved Escherichia coli cells by seminalplasmin, an antimicrobial protein from bovine seminal plasma

Abstract A pair of relA ⁺ and relA E. coli strains, otherwise isogenic, were studied with regard to the susceptibility of starved cells to lysis induced by the natural peptide seminalplasmin. Starved relA cells were more sensitive to seminalplasmin-induced lysis when compared to starved relA ⁺ cells. Nevertheless, pronounced lysis of starved relA ⁺ cells was observed with increase in the concentration of seminalplasmin. In conctrast, ampicillin could not lyse starved relA ⁺ cells even at very high concentrations. Further, seminalplasmin could cause loss of viability and degradation of peptidoglycan in starved relA ⁺ cells. These observations suggest that, unlike many other antibiotics, seminalplasmin can induce autolysis under the conditions of a stringent response.     

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.