RESUMO
Galactose is an abundant monosaccharide found exclusively in mammals as galactopyranose (Gal p), the six-membered ring form of this sugar. In contrast, galactose appears in many pathogenic microorganisms as the five-membered ring form, galactofuranose (Gal f). Gal f biosynthesis begins with the conversion of UDP-Gal p to UDP-Gal f catalyzed by the flavoenzyme UDP-galactopyranose mutase (UGM). Because UGM is essential for the survival and proliferation of several pathogens, there is interest in understanding the catalytic mechanism to aid inhibitor development. Herein, we have used kinetic measurements and molecular dynamics simulations to explore the features of UGM that control the rate-limiting step (RLS). We show that UGM from the pathogenic fungus Aspergillus fumigatus also catalyzes the isomerization of UDP-arabinopyranose (UDP-Ara p), which differs from UDP-Gal p by lacking a -CH2-OH substituent at the C5 position of the hexose ring. Unexpectedly, the RLS changed from a chemical step for the natural substrate to product release with UDP-Ara p. This result implicated residues that contact the -CH2-OH of UDP-Gal p in controlling the mechanistic path. The mutation of one of these residues, Trp315, to Ala changed the RLS of the natural substrate to product release, similar to the wild-type enzyme with UDP-Ara p. Molecular dynamics simulations suggest that steric complementarity in the Michaelis complex is responsible for this distinct behavior. These results provide new insight into the UGM mechanism and, more generally, how steric factors in the enzyme active site control the free energy barriers along the reaction path.
Assuntos
Aspergillus fumigatus/enzimologia , Transferases Intramoleculares/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/química , Aspergillus fumigatus/metabolismo , Cristalografia por Raios X , Galactose/análogos & derivados , Galactose/metabolismo , Humanos , Transferases Intramoleculares/química , Isomerismo , Cinética , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato , Difosfato de Uridina/análogos & derivados , Difosfato de Uridina/metabolismo , Uridina Difosfato Galactose/metabolismo , Açúcares de Uridina Difosfato/metabolismoRESUMO
The synthesis of noncellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as a substrate. We have cloned and characterized a nucleotide sugar transporter from Arabidopsis thaliana (L.) Heynh. named AtUTr2. Expression in tobacco and Saccharomyces cerevisiae and subsequent biochemical characterization indicate that AtUTr2 transports UDP-galactose, but not UDP-glucose, UDP-N-acetyl glucosamine, UDP-xylose, UDP-glucuronic acid, GDP-fucose or GDP-mannose. Experiments expressing an AtUTr2-GFP fusion protein in onion epidermal cells suggest that AtUTr2 is located in the Golgi apparatus. Finally, northern analysis indicates that the AtUTr2 transcript was more abundant in roots and calli although it was also present in other Arabidopsis organs but at lower levels. Therefore, AtUTr2 is a nucleotide sugar transporter capable of transporting UDP-galactose that may play an important role in the synthesis of galactose-containing glycoconjugates in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Uridina Difosfato Galactose/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Filogenia , Transporte Proteico , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Nicotiana/genética , Uridina Difosfato Glucose/metabolismoRESUMO
The synthesis of non-cellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as substrate. The topology of these reactions is not known, although the orientation of a plant galactosyltransferase involved in the biosynthesis of galactomannans in fenugreek is consistent with a requirement for UDP-galactose in the lumen of the Golgi cisternae. Here we provide evidence that sealed, right-side-out Golgi vesicles isolated from pea stems transport UDP-galactose into their lumen and transfer galactose, likely to polysaccharides and other acceptors. In addition, we identified and cloned AtUTr1, a gene from Arabidopsis thaliana that encodes a multitransmembrane hydrophobic protein similar to nucleotide sugar transporters. Northern analysis showed that AtUTr1 is indeed expressed in Arabidopsis. AtUTr1 is able to complement the phenotype of MDCK ricin-resistant cells; a mammalian cell line deficient in transport of UDP-galactose into the Golgi. In vitro assays using a Golgi-enriched vesicle fraction obtained from Saccharomyces cerevisiae expressing AtUTr1-MycHis is able to transport UDP-galactose but also UDP-glucose. AtUTr1- MycHis does not transport GDP-mannose, GDP-fucose, CMP-sialic acid, UDP-glucuronic acid, or UDP-xylose when expressed in S. cerevisiae. AtUTr1 is the first transporter described that is able to transport UDP-galactose and UDP-glucose. Thus AtUTr1 may play an important role in the synthesis of glycoconjugates in Arabidopsis that contain galactose and glucose.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte de Monossacarídeos/química , Uridina Difosfato Galactose/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Northern Blotting , Linhagem Celular , Clonagem Molecular , DNA Complementar/metabolismo , Cães , Genoma de Planta , Complexo de Golgi/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana Transportadoras/metabolismo , Microssomos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Pisum sativum , Filogenia , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Uridina Difosfato Glucose/metabolismoRESUMO
Previous studies from this laboratory have shown that synthesis of GT3, the precursor of c series gangliosides, occurs in proximal Golgi compartments, as has been shown for the synthesis of GM3 and GD3, the precursors of a and b series gangliosides, respectively. In this work we studied whether the synthesis of GM3, GD3, and GT3 occurs in the same or in different compartments of the proximal Golgi. For this, we examined in retina cells (a) the effect of monensin, a sodium ionophore that affects mostly the trans Golgi and the trans Golgi network function, on the metabolic labeling of glycolipids from [3H]Gal by cultured cells from 7- and 10-day chick embryos and (b) the labeling in vitro of endogenous glycolipids of Golgi membrane preparations from 7-day embryos incubated with UDP-[3H]Gal. In (a), 1 microM monensin produced a twofold accumulation of radioactive glucosylceramide and a decrease to approximately 50 and 20% of total ganglioside labeling in 7- and 10-day cells, respectively. At both ages, monensin produced a threefold accumulation of radioactive GM3 and an inhibition of > 90% of GT3, GM1, GD1a, and GT1b synthesis. GD3 synthesis was inhibited approximately 30 and 70%, respectively, in 7- and 10-day cells. In (b), > 80% of the [3H]Gal was incorporated into endogenous glucosylceramide to form radioactive lactosylceramide. About 90% of [3H]Gal-labeled lactosylceramide was converted into GM3, and most of this in turn into GD3 when unlabeled CMP-NeuAc was also present in the incubation system. Under the same conditions, however, < 5% of labeled GD3 was converted into GT3. Golgi membranes incubated with CMP-[3H]NeuAc incorporated approximately 20% of [3H]NeuAc into endogenous GT3, and this percentage was not affected by 1 microM monensin. These results indicate that synthesis of GT3 is carried out in a compartment of the proximal Golgi different from those for lactosylceramide, GM3, and GD3 synthesis. Results from the experiments with monensin point to the cis/medial Golgi as the main compartment for coupled synthesis of lactosylceramide, GM3, and GD3 and to the trans Golgi as the main compartment for synthesis of GT3.
Assuntos
Gangliosídeo G(M3)/biossíntese , Gangliosídeos/biossíntese , Complexo de Golgi/metabolismo , Lactosilceramidas/biossíntese , Células Ganglionares da Retina/metabolismo , Animais , Sequência de Carboidratos , Células Cultivadas , Embrião de Galinha , Gangliosídeos/química , Gangliosídeos/isolamento & purificação , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Dados de Sequência Molecular , Monensin/farmacologia , Uridina Difosfato Galactose/metabolismoRESUMO
Impurities in a reagent dehydrogenase caused overestimates of erythrocytic uridine diphosphate glucose and accounted for clinically important differences in results between those of one group of investigators using enzymatic methods and those of two other groups using enzymatic methods, high-performance liquid chromatography, and nuclear magnetic resonance. These data have relevance for the current debate regarding the pathophysiologic changes in galactosemia.