Characterization of Ligand Binding to the Bifunctional Key Enzyme in the Sialic Acid Biosynthesis by NMR

Blume, Astrid; Benie, Andrew J.; Stolz, Florian; Schmidt, Richard R.; Reutter, Werner; Hinderlich, Stephan; Peters, Thomas

doi:10.1074/jbc.m410238200

Cited by 24 publications

(27 citation statements)

References 43 publications

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“…As in other nucleotide sugar binding enzymes (e.g. UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase) (43)(44)(45), the nucleotide moiety receives the largest amount of saturation transfer. In particular, H1 of the ribose and H5 of the uridine ring are in intimate contact with the UGP protein.…”

Section: Discussionmentioning

confidence: 99%

Molecular Cloning of the Leishmania major UDP-glucose Pyrophosphorylase, Functional Characterization, and Ligand Binding Analyses Using NMR Spectroscopy

Lamerz

Haselhorst

Bergfeld

et al. 2006

Journal of Biological Chemistry

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The dense glycocalyx surrounding the protozoan parasite Leishmania is an essential virulence factor. It protects the parasite from hostile environments in the sandfly vector and mammalian host and supports steps of development and invasion. Therefore, new therapeutic concepts concentrate on disturbing glycocalyx biosynthesis. Deletion of genes involved in the metabolism of galactose and mannose have been shown to drastically reduce Leishmania virulence. Here we report the identification of Leishmania major UDP-glucose pyrophosphorylase (UGP). UGP catalyzes the formation of UDP-glucose from glucose 1-phosphate and UTP. This activation step enables glucose to enter metabolic pathways and is crucial for the activation of galactose. UDP-galactose is made from UDP-glucose by nucleotide-donor transfer to galactose 1-phosphate or by epimerization of the glucose moiety. Isolated in a complementation cloning approach, the activity of L. major UGP was proven in vitro. Moreover, purified protein was used to investigate enzyme kinetics, quaternary organization, and binding of ligands. Whereas sequestration by oligomerization is a known regulatory mechanism for eukaryotic UGPs, the recombinant as well as native L. major UGP migrated as monomer in size exclusion chromatography and in accord with this showed simple Michaelis-Menten kinetics toward all substrates. In saturation transfer difference (STD)-NMR studies, we clearly demonstrated that the molecular geometry at position 4 of glucose is responsible for substrate specificity. Furthermore, the ␥-phosphate group of UTP is essential for binding and for induction of the open conformation, which then allows entry of glucose 1-phosphate. Our data provide the first direct proof for the ordered bi-bi mechanism suggested in earlier studies.Leishmania are protozoan parasites that cause severe diseases in human and animals. Depending on the Leishmania strain, disease manifestations can range from self-healing cutaneous lesions to fatal visceral forms (1). Based on records of the World Health Organization, 12 million people are infected worldwide, but no vaccine or specific drug is available to prevent or treat Leishmaniasis.Leishmania are covered by a dense glycoconjugate layer, the glycocalyx, which helps the parasite to withstand hostile environments in the sandfly vector and mammalian host. Moreover, structural changes in glycocalyx components accompany the life cycle of the parasites and are crucial for the transmission of the parasite (2, 3).A major component of the glycoconjugates is galactose, which is dynamically added in the Golgi apparatus. Both transport of galactose to the Golgi lumen and insertion into glycoconjugates depend on the synthesis of the nucleotide sugar UDP-Gal in the cytoplasm of the cell. Whereas synthesis of most nucleotide sugars is catalyzed by pyrophosphorylases using the respective nucleotide triphosphate as substrate (for a review, see Ref. 4), activation of galactose is achieved by nucleotide exchange between UDP-Glc and galactose 1-phosphate (Gal-...

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Section: Discussionmentioning

confidence: 99%

Molecular Cloning of the Leishmania major UDP-glucose Pyrophosphorylase, Functional Characterization, and Ligand Binding Analyses Using NMR Spectroscopy

Lamerz

Haselhorst

Bergfeld

et al. 2006

Journal of Biological Chemistry

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“…The site-directed mutagenesis of the DDxxD motif in each domain further indicated that the 92 DDVTD motif and 474 DDFQD motif were part of the active site for the terpene cyclase and prenyltransferase reactions, respectively. To date, several classes of multifunctional enzymes have been reported in the biosynthesis of natural products such as polyketides, terpenes, and primary metabolites (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). These enzymes mechanically catalyze distinct reactions by possessing multiple enzyme domains.…”

Section: Discussionmentioning

confidence: 99%

Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi

Toyomasu

Tsukahara

Kaneko

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

191

186

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Fusicoccins are a class of diterpene glucosides produced by the plant-pathogenic fungus Phomopsis amygdali. As modulators of 14-3-3 proteins, fusicoccins function as potent activators of plasma membrane H ؉ -ATPase in plants and also exhibit unique biological activity in animal cells. Despite their well studied biological activities, no genes encoding fusicoccin biosynthetic enzymes have been identified. Cyclic diterpenes are commonly synthesized via cyclization of a C20 precursor, geranylgeranyl diphosphate (GGDP), which is produced through condensation of the universal C5 isoprene units dimethylallyl diphosphate and isopentenyl diphosphate by prenyltransferases. We found that (؉)-fusicocca-2,10 (14)-diene, a tricyclic hydrocarbon precursor for fusicoccins, is biosynthesized from the C5 isoprene units by an unusual multifunctional enzyme, P. amygdali fusicoccadiene synthase (PaFS), which shows both prenyltransferase and terpene cyclase activities. The functional analysis of truncated mutants and site-directed mutagenesis demonstrated that PaFS consists of two domains: a terpene cyclase domain at the N terminus and a prenyltransferase domain at the C terminus. These findings suggest that fusicoccadiene can be produced efficiently in the fungus by using the C5 precursors, irrespective of GGDP availability. In fact, heterologous expression of PaFS alone resulted in the accumulation of fusicocca-2,10 (14)-diene in Escherichia coli cells, whereas no product was detected in E. coli cells expressing Gibberella fujikuroi ent-kaurene synthase, another fungal diterpene cyclase that also uses GGDP as a substrate but does not contain a prenyltransferase domain. Genome walking suggested that fusicoccin biosynthetic enzymes are encoded as a gene cluster near the PaFS gene.biosynthesis ͉ cyclase ͉ prenyltransferase F usicoccins (FC) A (1, 2) and J (3, 4) ( Fig. 1) are the major metabolites produced by the plant-pathogenic fungus Phomopsis (Fusicoccum) amygdali (Del.) (5). Fusicoccin A and the structurally related cotylenins (CN) (6-8), e.g., cotylenin A (Fig.

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“…The obtained results indicated the strong binding of caged nucleotides to the investigated proteins. Similarly, Blume et al [57] used the same technique to study the interaction of the epimerase site of the bifunctional enzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase with its natural substrate, UDP-N-acetylglucosamine (UDP-GlcNAc) and derivatives. STD-NMR allowed for ligand epitope mapping and the determination of ligand binding affinity.…”

Section: Ligand-protein Interactionsmentioning

confidence: 99%

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

Novak¹,

Jednačak²

2012

Physico-Chemical Methods in Drug Discovery and Development

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Characterization of Ligand Binding to the Bifunctional Key Enzyme in the Sialic Acid Biosynthesis by NMR

Cited by 24 publications

References 43 publications

Molecular Cloning of the Leishmania major UDP-glucose Pyrophosphorylase, Functional Characterization, and Ligand Binding Analyses Using NMR Spectroscopy

Molecular Cloning of the Leishmania major UDP-glucose Pyrophosphorylase, Functional Characterization, and Ligand Binding Analyses Using NMR Spectroscopy

Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

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