Structural Basis for Subunit Assembly in UDP-glucose Pyrophosphorylase from Saccharomyces cerevisiae

Roeben, Annette; Plitzko, Jürgen M.; Körner, Roman; Böttcher, Ulrike M K; Siegers, Katja; Hayer‐Hartl, Manajit; Bracher, A.

doi:10.1016/j.jmb.2006.08.079

Cited by 47 publications

(79 citation statements)

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“…In contrast to the monomeric L. major and plant UGPases (8, 11) the active ani- mal and fungal UGPases are oligomeric. The enzyme, Ugp1p from Saccharomyces cerevisiae forms octamers in crystals (29). This enzyme also harbors a C-terminal domain with a lefthanded parallel ␤-helix that mediates the association in the homooctamer.…”

Section: Discussionmentioning

confidence: 99%

Open and Closed Structures of the UDP-glucose Pyrophosphorylase from Leishmania major

Steiner

Lamerz

Hess

et al. 2007

Journal of Biological Chemistry

117

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Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel ␤-helix (L␤H), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.Uridinediphosphate-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) 2 is present in all three kingdoms of life and catalyzes the reaction of UTP ϩ glucose 1-phosphate 3 UDP-glucose ϩ PP i in the presence of Mg 2ϩ in vivo. UDP-glucose, the activated form of glucose, plays an essential role in the metabolism of carbohydrates in all organisms. UDP-glucose is the main glucosyl donor for the formation of glycogen, starch, and cellulose, as well as for the synthesis of glucose-containing glycolipids, glycoproteins, and proteoglycans (1, 2). In addition, other important nucleotide sugars such as UDP-xylose, UDP-glucuronic acid, and UDP-galactose are derived from UDP-glucose. In bacteria some of these activated sugars are used to build the bacterial polysaccharide capsule that often represents the sole determinant of virulence of these organisms. In Streptococcus pneumoniae, for example, it was known that mutants containing an inactivated UGPase gene (galU) are completely avirulent, as they are unable to form the polysaccharide capsule (3). Similarly, UGPase is involved in the biosynthesis of the lipopolysaccharide core in Escherichia coli, resulting in a reduced adhesion behavior of E. coli galU mutants (4).The protozoan parasite Leishmania is the causative agent of a widespread group of diseases collectively known as Leishmaniasis. The disease affects more than 12 million people worldwide and until now there is no specific drug available to cure the disease.3 Leishmania express various glycoconjugates on their cell surface that is dynamically modified during the parasite life cycle allowing the survival and proliferation in the sand fly vector as well as in the mammalian host (6, 7).The biosynthesis of glycoconjugates essentially depends on the availability of activated nucleotide sugars. UGPase represents a key position in...

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Section: Discussionmentioning

confidence: 99%

Open and Closed Structures of the UDP-glucose Pyrophosphorylase from Leishmania major

Steiner

Lamerz

Hess

et al. 2007

Journal of Biological Chemistry

117

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“…The b-sheet of the Rossmann fold is a structural basis for the active site of the enzyme. This general structural blueprint is shared by other UDP-Glc-producing pyrophosphorylases (UGPase-A, UGPase-B, and UAGPase; Peneff et al, 2001;Geisler et al, 2004;Roeben et al, 2006;Maruyama et al, 2007;McCoy et al, 2007;Steiner et al, 2007;Okazaki et al, 2009;Kleczkowski et al, 2010;. Differences concern mostly details of the C-terminal domain, which, in USPase, is composed of two parts: a distorted b-sheet resembling that in the C-terminal domain of human AGX (UAGPase), and a left-handed b-helix that is characteristic of the C-terminal domain of eukaryotic UGPase-A.…”

Section: Protein Structurementioning

confidence: 95%

UDP-Sugar Pyrophosphorylase: A New Old Mechanism for Sugar Activation

2011

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“…7). On the other hand, the C94S, C191S, and C354S mutant enzymes behaved [36]; 2i5k: UDP-GlcPPase from Saccharomyces cerevisiae [37]; 2icy: UDP-GlcPPase from Arabidopsis thaliana with bound UDP-Glc [36]; 2icx: UDP-GlcPPase from Arabidopsis thaliana with bound UTP [36]; 2oeg: UDP-GlcPPase from Leishmania major [5]. as the wild type protein when faced with any of the three oxidative treatments (Fig.…”

Section: Characterization Of Methionyl and Cysteinyl Mutant Enzymesmentioning

confidence: 98%

“…Building of the structure model of EhiUDP-GlcPPase was based in templates detailed in Table 3; derived from the known X-ray structure of the homologous enzyme from eukaryotes [5,36,37]. Almost all selected templates have significant number of identical residues (ca.…”

Section: Molecular Modelingmentioning

confidence: 99%

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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Structural Basis for Subunit Assembly in UDP-glucose Pyrophosphorylase from Saccharomyces cerevisiae

Cited by 47 publications

References 50 publications

Open and Closed Structures of the UDP-glucose Pyrophosphorylase from Leishmania major

Open and Closed Structures of the UDP-glucose Pyrophosphorylase from Leishmania major

UDP-Sugar Pyrophosphorylase: A New Old Mechanism for Sugar Activation

Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

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