RESUMO
The Leishmania parasite glycocalyx is rich in galactose-containing 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.
Assuntos
Galactosefosfatos/metabolismo , Leishmania major/enzimologia , Nucleotidiltransferases/metabolismo , Proteínas de Protozoários/metabolismo , Difosfato de Uridina/metabolismo , Sequência de Aminoácidos , Animais , Evolução Molecular , Galactosefosfatos/genética , Glucofosfatos/genética , Glucofosfatos/metabolismo , Glicocálix/enzimologia , Glicocálix/genética , Leishmania major/genética , Dados de Sequência Molecular , Nucleotidiltransferases/genética , Proteínas de Protozoários/genética , Especificidade por Substrato/fisiologia , Difosfato de Uridina/genética , Uridina Trifosfato/genética , Uridina Trifosfato/metabolismoRESUMO
The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose (UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:alpha-D-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis.
Assuntos
Leishmania major/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/genética , Uridina Difosfato Galactose/metabolismo , Uridina Difosfato Glucose/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Leishmania major/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Deleção de Sequência , Transdução de Sinais , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Uridina Difosfato Galactose/química , Uridina Difosfato Glucose/químicaRESUMO
Saturation transfer difference (STD) Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful method for studying protein-ligand interactions in solution. The STD NMR method is capable of identifying the binding epitope of a ligand when bound to its receptor protein. Ligand protons that are in close contact with the receptor protein receive a higher degree of saturation, and as a result stronger STD NMR signals can be observed. Protons that are either less or not involved in the binding process reveal no STD NMR signals. Therefore, the STD NMR method is an excellent tool to investigate how a binding ligand interacts with its receptor molecule. The STD NMR experiment is easy to implement and only small amounts of native protein are required. This chapter comprises a detailed experimental protocol to acquire STD NMR spectra and determine the binding epitope of a ligand bound to its target protein. As representative examples the ligands uridyl-triphosphate (UTP) and uridyl-glucose-diphosphate (UDP-glucose) when bound to the Leishmania major UDP-glucose-pyrophosphorylase (UGP) as target protein are examined.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/metabolismo , Soluções/farmacologia , Animais , Sítios de Ligação , Sequência de Carboidratos , Carboidratos/análise , Carboidratos/química , Humanos , Modelos Biológicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Soluções/químicaRESUMO
Nucleotide sugars and the enzymes that are responsible for their synthesis are indispensable for the production of complex carbohydrates and, thus, for elaboration of a protective cellular coat for many organisms such as the protozoan parasite Leishmania. These activated sugars are synthesized de novo or derived from salvaged monosaccharides. In addition to UDP-glucose (UDP-Glc) pyrophosphorylase, which catalyzes the formation of UDP-Glc from substrates UTP and glucose-1-phosphate, Leishmania major and plants express a UDP-sugar pyrophosphorylase (USP) that exhibits broad substrate specificity in vitro. The enzyme, likely involved in monosaccharide salvage, preferentially generates UDP-Glc and UDP-galactose, but it may also activate other hexose- or pentose-1-phosphates such as galacturonic acid-1-phosphate or arabinose-1-phosphate. In order to gain insight into structural features governing the differences in substrate specificity, we determined the crystal structure of the L. major USP in the APO-, UTP-, and UDP-sugar-bound conformations. The overall tripartite structure of USP exhibits a significant structural homology to other nucleotidyldiphosphate-glucose pyrophosphorylases. The obtained USP structures reveal the structural rearrangements occurring during the stepwise binding process of the substrates. Moreover, the different product complexes explain the broad substrate specificity of USP, which is enabled by structural changes in the sugar binding region of the active site.
Assuntos
Glucofosfatos/metabolismo , Leishmania major/enzimologia , Fosfatos Açúcares/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/química , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Açúcares de Uridina Difosfato/metabolismo , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
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 beta-helix (LbetaH), 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.
Assuntos
Leishmania major/enzimologia , Dobramento de Proteína , Proteínas de Protozoários/química , UTP-Glucose-1-Fosfato Uridililtransferase/química , Fatores de Virulência/química , Animais , Apoenzimas/química , Apoenzimas/metabolismo , Catálise , Cristalografia por Raios X , Glicoconjugados/biossíntese , Glicoconjugados/química , Leishmania major/patogenicidade , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Fatores de Virulência/metabolismoRESUMO
Considering the high incidence of galactofuranose (Gal(f)) in pathogens and its absence from higher eukaryotes, the enzymes involved in the biosynthesis of this unusual monosaccharide appear as attractive drug targets. However, although the importance of Gal(f) in bacterial survival or pathogenesis is established, its role in eukaryotic pathogens is still undefined. Recently, we reported the identification and characterization of the first eukaryotic UDP-galactopyranose mutases. This enzyme holds a central role in Gal(f) metabolism by providing UDP-Gal(f) to all galactofuranosyltransferases. In this work, the therapeutical potential of Gal(f) metabolism in Leishmania major was hence evaluated by targeted replacement of the GLF gene encoding UDP-galactopyranose mutase. In L. major, Gal(f) is present in the membrane anchor of the lipophosphoglycan (LPG) and in glycoinositolphospholipids. Accordingly, the generated glf(-) mutant is deficient in LPG backbone and expresses truncated glycoinositolphospholipids. These structural changes do not influence the in vitro growth of the parasite but lead to an attenuation of virulence comparable with that observed with a mutant exclusively deficient in LPG.
Assuntos
Deleção de Genes , Transferases Intramoleculares/deficiência , Leishmania major/genética , Leishmania major/patogenicidade , Proteínas de Protozoários/genética , Animais , Galactose/análogos & derivados , Galactose/metabolismo , Glicolipídeos/biossíntese , Glicolipídeos/genética , Transferases Intramoleculares/metabolismo , Leishmania major/enzimologia , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/enzimologia , Leishmaniose Cutânea/genética , Fosfolipídeos/biossíntese , Fosfolipídeos/genética , Proteínas de Protozoários/metabolismo , Difosfato de Uridina/análogos & derivados , Difosfato de Uridina/metabolismoRESUMO
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 gamma-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.