UDPglucose pyrophosphorylase from Xanthomonas spp. Characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation

Bosco, María Belén; Machtey, Matías; Iglesias, Alberto A.; Aleanzi, Mabel Cristina

doi:10.1016/j.biochi.2008.09.001

Cited by 27 publications

(40 citation statements)

References 44 publications

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“…Also concordant with previous reports were hyperbolic kinetics obtained for Glc1P and PPi, and sigmoidal kinetics for the substrates UTP and UDP-Glc; data resulting characteristic for these kinds of enzymes. Different to that found for UDPGlcPPases from bacteria [6], the entamoebic enzyme showed no activity with other nucleotides: ATP, GTP or TTP.…”

Section: Discussioncontrasting

confidence: 96%

“…As a whole, these results on specificity are in agreement with data previously reported for other UDP-GlcPPases [6e8], except for the use of TTP as a substrate by the enzyme from Xanthomonas spp. [6]. Moreover, the recombinant enzyme was insensitive to regulation by any allosteric effector.…”

Section: Expression and Properties Of Recombinant Ehiudp-glcppasementioning

confidence: 97%

“…The values of activity in the forward direction thus obtained is similar to that previously found for the enzyme from Xanthomonas spp. [6]. The reaction catalyzed by pyrophosphorylases is freely reversible in vitro, with an equilibrium close to 1; although differences were found respect to rates of the forward or reverse reaction [26], which could be attributed to regulation or characteristics associated with protein oligomerization [6,27].…”

Section: Expression and Properties Of Recombinant Ehiudp-glcppasementioning

confidence: 97%

“…[6]. The reaction catalyzed by pyrophosphorylases is freely reversible in vitro, with an equilibrium close to 1; although differences were found respect to rates of the forward or reverse reaction [26], which could be attributed to regulation or characteristics associated with protein oligomerization [6,27]. Data in Table 2 indicate that the EhiUDP-GlcPPase exhibited lower V max and affinity for substrates than the enzyme from L. major [5,7], Arabidopsis thaliana [28], and barley [29]; whereas kinetics for the recombinant enzyme are similar than those reported for one specific UDP-GlcPPase partially purified from E. histolytica [30], except for a lower affinity of the former for UTP.…”

Section: Expression and Properties Of Recombinant Ehiudp-glcppasementioning

confidence: 99%

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Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

et al. 2011

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a b s t r a c tAmoebiasis 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 UDPglucose 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.

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Section: Discussioncontrasting

confidence: 96%

Section: Expression and Properties Of Recombinant Ehiudp-glcppasementioning

confidence: 97%

Section: Expression and Properties Of Recombinant Ehiudp-glcppasementioning

confidence: 97%

Section: Expression and Properties Of Recombinant Ehiudp-glcppasementioning

confidence: 99%

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Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

et al. 2011

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“…Nucleotide metabolism produces the required nucleotides for viral genome replication, which was shown to be crucial during T7 infection (Qimron et al, 2006). UDP-glucose and UDP-galactose are key intermediates for the production of oligo-and polysaccharides (Bosco et al, 2009;Ebrecht et al, 2015). For example, the nucleotide sugars (UDP-glucose, UDP-alpha-DManNAc3NAcA and UDP-alpha-D-GlcNAc3NAcA) are precursors for lipopolysaccharide biosynthesis .…”

Section: Resultsmentioning

confidence: 99%

High coverage metabolomics analysis reveals phage-specific alterations to Pseudomonas aeruginosa physiology during infection

et al. 2016

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Phage-mediated metabolic changes in bacteria are hypothesized to markedly alter global nutrient and biogeochemical cycles. Despite their theoretic importance, experimental data on the net metabolic impact of phage infection on the bacterial metabolism remains scarce. In this study, we tracked the dynamics of intracellular metabolites using untargeted high coverage metabolomics in Pseudomonas aeruginosa cells infected with lytic bacteriophages from six distinct phage genera. Analysis of the metabolomics data indicates an active interference in the host metabolism. In general, phages elicit an increase in pyrimidine and nucleotide sugar metabolism. Furthermore, clear phage-specific and infection stage-specific responses are observed, ranging from extreme metabolite depletion (for example, phage YuA) to complete reorganization of the metabolism (for example, phage phiKZ). As expected, pathways targeted by the phage-encoded auxiliary metabolic genes (AMGs) were enriched among the metabolites changing during infection. The effect on pyrimidine metabolism of phages encoding AMGs capable of host genome degradation (for example, YuA and LUZ19) was distinct from those lacking nuclease-encoding genes (for example, phiKZ), which demonstrates the link between the encoded set of AMGs of a phage and its impact on host physiology. However, a large fraction of the profound effect on host metabolism could not be attributed to the phage-encoded AMGs. We suggest a potentially crucial role for small, 'non-enzymatic' peptides in metabolism take-over and hypothesize on potential biotechnical applications for such peptides. The highly phage-specific nature of the metabolic impact emphasizes the potential importance of the 'phage diversity' parameter when studying metabolic interactions in complex communities.

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The galU gene of Xanthomonas campestris pv. campestris is involved in bacterial attachment, cell motility, polysaccharide synthesis, virulence, and tolerance to various stresses

Liao

et al. 2014

Arch Microbiol

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Uridine triphosphate (UTP)-glucose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.9) is an enzyme that catalyzes the formation of uridine diphosphate (UDP)-glucose from UTP and glucose-1-phosphate. GalU is involved in virulence in a number of animal-pathogenic bacteria since its product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharide and exopolysaccharide. However, its function in Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, is unclear. Here, we characterized a galU mutant of X. campestris pv. campestris and showed that the X. campestris pv. campestris galU mutant resulted in a reduction in virulence on the host cabbage. We also demonstrated that galU is involved in bacterial attachment, cell motility, and polysaccharide synthesis. Furthermore, the galU mutant showed increased sensitivity to various stress conditions including copper sulfate, hydrogen peroxide, and sodium dodecyl sulfate. In addition, mutation of galU impairs the expression of the flagellin gene fliC as well as the attachment-related genes xadA, fhaC, and yapH. In conclusion, our results indicate involvement of galU in the virulence factor production and pathogenicity in X. campestris pv. campestris, and a role for galU in stress tolerance of this crucifer pathogen.

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UDPglucose pyrophosphorylase from Xanthomonas spp. Characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation

Cited by 27 publications

References 44 publications

Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

High coverage metabolomics analysis reveals phage-specific alterations to Pseudomonas aeruginosa physiology during infection

The galU gene of Xanthomonas campestris pv. campestris is involved in bacterial attachment, cell motility, polysaccharide synthesis, virulence, and tolerance to various stresses

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