Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

Abstract

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.

Oligomerization, Membrane Association, and in Vivo Phosphorylation of Sugarcane UDP-glucose Pyrophosphorylase

“…The redox assay showed that the oxidized agent H 2 O 2 inactivated ScUGPase-1, and this inhibition was completely reversed by the addition of the reducing agent DTT. This result agrees with the previous report demonstrating that the activity of UGPase from E. histolytica is modulated by redox mechanisms (47). Reactive oxygen species, which include hydrogen peroxide (H 2 O 2 ), play crucial roles in modulating many physiologic and pathologic processes in vivo.…”

“…2B indicates the presence of a putative ScUGPase-1 dimer in sugarcane leaves. In addition, oxidizing agents regulate the quaternary state of UGPase (47). Therefore, we evaluated the range of levels of H 2 O 2 internode and leaf tissues of sugarcane.…”

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

“…For calculation of percentage of remaining activity, 100% was considered as the activity when the enzyme was incubated under the same conditions but in the absence of oxidants. Oxidation kinetics was analyzed as described elsewhere [6,28]. Briefly, the first order rate constant (k app ) for each oxidant concentration was calculated by fitting the curves to the equation: log relative activity = 2 − k app * t, using Origin™ 8.0 software.…”

“…Before use, TcrTRX and TcrTXN were reduced by incubation in buffer 100 mM MOPS-HCl pH 8.0, 5% (v/v) glycerol, 0.1 mM EDTA, and 0.5 mM DTT during 30 min at 25°C. Thiol proteins used for assays were purified according to protocols previously specified [6,29].…”

“…Although it is ubiquitously distributed in nature (including animals, plants and microorganisms) [1][2][3], the enzyme from eukaryotes is remarkably unrelated with that found in prokaryotes and they are not homologous proteins [2,4,5]. Recently [6], it has been demonstrated that the UDP-Glc PPase from Entamoeba histolytica is finely regulated by post-translational redox modification elicited by critical metabolites of the intracellular environment. The regulation is determinant for the distribution of carbohydrates into different metabolic fates.…”