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

Ebrecht, Ana Cristina; Diez, Matías Damián Asención; Piattoni, Claudia Vanesa; Guerrero, Sandra; Iglesias, Alberto A.

doi:10.1016/j.bbagen.2014.10.002

Cited by 16 publications

(19 citation statements)

References 69 publications

(134 reference statements)

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“…The oligomerization is regulated by subtle changes in hydrophobicity of an immediate environment, protein crowding, and the availability of substrates of UGPase (Kleczkowski et al ., , ; Decker et al ., ). Other levels of regulation, although still to be explored in more detail, involve redox modification (Soares et al ., ; Ebrecht et al ., ) and phosphorylation (Soares et al ., ). For USPase, there have been fewer reports on its regulation, but this could be related to the relative ‘novelty’ of this protein, first time identified in 2004 (Kotake et al ., ).…”

Section: Discussionmentioning

confidence: 99%

“…Whereas UGPases and USPases, based on their overall amino acid sequences, share less than 20% identity, the architecture of their active sites and the positioning of substrate binding domains are generally similar (Kleczkowski et al, 2011b). It is still surprising though, given that UGPase reacts only with Glc-1-P, and to some extent Gal-1-P (Decker et al, 2012;Ebrecht et al, 2015), whereas USPase can easily accommodate those and several other sugar-1-phosphates as substrates (Kotake et al, 2004(Kotake et al, , 2007Litterer et al, 2006a;Damerow et al, 2010;Yang and Bar-Peled, 2010). The identified five inhibitors of barley UGPase and Leishmania USPase were also effective against activities of purified Arabidopsis USPase and two isozymes of Arabidopsis UGPase (UGPase1 and UGPase2).…”

Section: Discussionmentioning

confidence: 99%

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Identification and characterization of inhibitors of UDP‐glucose and UDP‐sugar pyrophosphorylases for in vivo studies

2017

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Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies.The Plant Journal, 90 (6) SUMMARY UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDPsugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 lM, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 lM. Their effects on pollen germination were relieved by addition of UDPglucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Identification and characterization of inhibitors of UDP‐glucose and UDP‐sugar pyrophosphorylases for in vivo studies

2017

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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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“…Holzl et al [42] found that glucosyl/galactosyltransferases from bacteria Agrobacterium tumefaciens and Mesorhizobium loti display multiple substrate specificities and can transfer three successive glucosyl- and galactosyl residues to diacylglycerol. Recently it has been reported that some giardial enzymes of carbohydrate metabolic pathways are promiscuous in nature and can utilize more than one substrate [43]. For instance, UDP-glucose pyrophosphorylase in Giardia can utilize galactose-1-phosphate.…”

Section: Discussionmentioning

confidence: 99%

“…For instance, UDP-glucose pyrophosphorylase in Giardia can utilize galactose-1-phosphate. This allows the cell to bypass several steps to produce UDP-Gal from galactose-1-phosphate because the genes for galactose transferase, galactose kinase, UDP-galactose pyrophosphatase, and phosphoglucomutase are not present in this parasite [43]. [ Likewise, pyridoxal phosphate-dependent alanine racemase shows promiscuity with cystathion β lyase and exhibits different Km and Kcat values.…”

Section: Discussionmentioning

confidence: 99%

Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation

Robles-Martinez¹,

Mendez

Apodaca

et al. 2017

Molecular and Biochemical Parasitology

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The stage differentiation from trophozoite to cyst (i.e., encystation) is an essential step for Giardia to survive outside its human host and spread the infection via the fecal-oral route. We have previously shown that Giardia expresses glucosylceramide transferase 1 (GlcT1) enzyme, the activity of which is elevated during encystation. We have also reported that blocking the activity of gGlcT1 interferes with the biogenesis of encystation-specific vesicles (ESVs) and cyst viability in Giardia. To further understand the role of this enzyme and how it regulates encystation, we overexpressed, knocked down, and rescued the giardial GlcT1 (gGlcT1) gene and measured its enzymatic activity in live parasites as well as in isolated membrane fractions using NBD-ceramide and UDP-glucose or UDP-galactose. We observed that gGlcT1 is able to catalyze the synthesis of both glucosylceramide (GlcCer) and galactosylceramide (GalCer), however the synthesis of GalCer is 2–3 fold higher than of GlcCer. Although both activities follow Michaelis–Menten kinetics, the bindings of UDP-glucose and UDP-galactose with the enzyme appear to be non-competitive and independent of each other. The modulation of gGlcT1 synthesis concomitantly influenced the expression cyst-wall protein (CWP) and overall encystation. We propose that gGlcT1 is a unique enzyme and that Giardia uses this enzyme to synthesize both GlcCer and GalCer to facilitate the process of encystation/cyst production.

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

Cited by 16 publications

References 69 publications

Identification and characterization of inhibitors of UDP‐glucose and UDP‐sugar pyrophosphorylases for in vivo studies

Identification and characterization of inhibitors of UDP‐glucose and UDP‐sugar pyrophosphorylases for in vivo studies

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

Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation

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