Oligomerization status, with the monomer as active species, defines catalytic efficiency of UDP-glucose pyrophosphorylase

Martz, Françoise; Wilczyńska, Małgorzata; Kleczkowski, Leszek A.

doi:10.1042/bj20020772

Cited by 69 publications

(83 citation statements)

References 32 publications

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“…Sequestration by oligomerization has recently been suggested to provide an important principle in the regulation of these enzymes (31). Asking whether the L. major enzyme is regulated in the same manner, we investigated the oligomerization status of recombinantly expressed L. major UGP.…”

Section: Major Ugp Is a Monomer With Simple Michaelis-menten Kinetmentioning

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: Major Ugp Is a Monomer With Simple Michaelis-menten Kinetmentioning

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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“…major USP Is a Monomer Exhibiting Broad Substrate Specificity-Oligomerization has been shown to regulate the activity of barley UGP (27) but not that of L. major UGP (22). We have thus investigated the oligomerization state of L. major C-terminally His-tagged USP (LmjUSP-His 6 ) expressed in E. coli BL21 (DE3) cells and purified to homogeneity via nickel affinity, anion-exchange, and size-exclusion chromatographies (Fig.…”

Section: Major Usp Is Involved In Biosynthesis Of Udp-glucose And/mentioning

confidence: 99%

Leishmania UDP-sugar Pyrophosphorylase

Damerow

Lamerz

Haselhorst

et al. 2010

Journal of Biological Chemistry

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The Leishmania parasite glycocalyx is rich in galactosecontaining glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

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“…Filters were washed and incubated with the horseradish peroxidase substrate (Immobilon western; Millipore) before exposure to film (Kodak). UGPase, a cytosolic protein (Martz et al, 2002), was detected with anti-UGPase rabbit polyclonal antibodies (Agrisera) diluted 1:3,000 followed by anti-rabbit alkaline phosphatase-conjugated antibodies diluted 1:5,000. Membranes were then submerged to luminol mix and incubate for 2 min followed by x-ray film exposure.…”

Section: Protein Extraction and Immunoblottingmentioning

confidence: 99%

SUMO Is a Critical Regulator of Salt Stress Responses in Rice

et al. 2016

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(A.S.).SUMO (Small Ubiquitin-like Modifier) conjugation onto target proteins has emerged as a very influential class of protein modification systems. SUMO1/2 double mutant plants are nonviable, underlining the importance of SUMO conjugation to plant survival. Once covalently bound, SUMO can alter a conjugated protein's stability and/or function. SUMO conjugation is a highly dynamic process that can be rapidly reversed by the action of SUMO proteases. The balance between the conjugated/ deconjugated forms is a major determinant in the modulation of SUMO-target function. Despite the important mechanistic role of SUMO proteases in model plants, until now the identity or the function of these regulatory enzymes has not been defined in any crop plant. In this report, we reveal the ubiquitin-like protease class of SUMO protease gene family in rice (Oryza sativa) and demonstrate a critical role for OsOTS1 SUMO protease in salt stress. OsOTS-RNAi rice plants accumulate high levels of SUMOconjugated proteins during salt stress and are highly salt sensitive; however, in non-salt conditions, they are developmentally indistinguishable from wild-type plants. Transgenic rice plants overexpressing OsOTS1 have increased salt tolerance and a concomitant reduction in the levels of SUMOylated proteins. We demonstrate that OsOTS1 confers salt tolerance in rice by increasing root biomass. High salinity triggers OsOTS1 degradation, indicating that increased SUMO conjugation in rice plants during salt stress is in part achieved by down-regulation of OTS1/2 activity. OsOTS1 is nuclear localized indicating a direct requirement of OsOTS1-dependent deSUMOylation activity in rice nuclei for salt tolerance.

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Oligomerization status, with the monomer as active species, defines catalytic efficiency of UDP-glucose pyrophosphorylase

Cited by 69 publications

References 32 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

Leishmania UDP-sugar Pyrophosphorylase

SUMO Is a Critical Regulator of Salt Stress Responses in Rice

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