guanosine-diphosphate-mannose and mannose-1-phosphate

Leptospira interrogans synthesizes a range of mannose-containing glycoconjugates relevant for its virulence. A prerequisite in the synthesis is the availability of the GDP-mannose, produced from mannose-1-phosphate and GTP in a reaction catalyzed by GDP-mannose pyrophosphorylase. The gene coding for a putative enzyme in L. interrogans was expressed in Escherichia coli BL21(DE3). The identity of this enzyme was confirmed by electrospray-mass spectroscopy, Edman sequencing and immunological assays. Gel filtration chromatography showed that the dimeric form of the enzyme is catalytically active and stable. The recombinant protein was characterized as a mannose-1-phosphate guanylyltransferase. S (0.5) for the substrates were determined both in GDP-mannose pyrophosphorolysis: 0.20 mM (GDP-mannose), 0.089 mM (PPi), and 0.47 mM; and in GDP-mannose synthesis: 0.24 mM (GTP), 0.063 mM (mannose-1-phosphate), and 0.45 mM (Mg(2+)). The enzyme was able to produce GDP-mannose, IDP-mannose, UDP-mannose and ADP-glucose. We obtained a structural model of the enzyme using as a template the crystal structure of mannose-1-phosphate guanylyltransferase from Thermus thermophilus HB8. Binding of substrates and cofactor in the model agree with the pyrophosphorylases reaction mechanism. Our studies provide insights into the structure of a novel molecular target, which could be useful for detection of leptospirosis and for the development of anti-leptospiral drugs.

Topics: Adenosine Diphosphate Glucose; Amino Acid Sequence; Bacterial Proteins; Blotting, Western; Chromatography, Gel; Guanosine Diphosphate Mannose; Leptospira interrogans; Mannosephosphates; Molecular Sequence Data; Nucleotidyltransferases; Protein Structure, Secondary; Sequence Homology, Amino Acid; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity; Uridine Diphosphate Sugars

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16-18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat/K m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.

Topics: Arabidopsis; Ascorbic Acid; Catalysis; Cell Death; Glycoproteins; Glycosylation; Guanosine Diphosphate Mannose; Hot Temperature; Mannosephosphates; Mutation; Phenotype; Phosphotransferases (Phosphomutases); Plant Proteins; Protein Disulfide-Isomerases; Recombinant Proteins; Seedlings

An open reading frame (ORF) encoding a putative GDP-mannose pyrophosphorylase (SsoGMPP) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino acid sequence homology to several archaeal, bacterial, and eukaryal GDP-mannose pyrophosphorylases such as guanidine diphosphomannose pyrophosphorylases (GMPPs) from Saccharomyces cerevisiae and Arabidopsis thaliana. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained as a fusion to glutathione S-transferase in Escherichia coli, under conditions suitable to reduce the formation of inclusion bodies. Specific assays performed at 60 degrees C revealed the presence of the archaeal synthesizing GDP-mannose enzyme activity in the cell extracts of the transformed E. coli. As a positive control, the same assays were performed at the mesophilic enzyme optimum temperature on the already characterized yeast recombinant GMPP. The recombinant protein was purified to homogeneity by glutathione sepharose affinity chromatography and its thermophilic nature could be verified. The enzyme was definitively identified by demonstrating its capability to catalyze also the reverse reaction of pyrophosphorolysis and, most interestingly, its high specificity for synthesizing GDP-mannose.

Topics: Amino Acid Sequence; Base Sequence; Catalysis; DNA, Archaeal; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Archaeal; Gene Expression Regulation, Enzymologic; Glutathione Transferase; Guanosine Diphosphate Mannose; Guanosine Triphosphate; Mannosephosphates; Molecular Sequence Data; Nucleotidyltransferases; Recombinant Fusion Proteins; RNA, Archaeal; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity; Sulfolobus

The enzyme that catalyzes the formation of GDP-d-mannose from GTP and alpha-d-mannose-1-P was purified about 2300-fold to near homogeneity from the soluble fraction of Mycobacterium smegmatis. At the final stage of purification, a major protein band of 37 kDa was observed and this band was specifically labeled, and in a concentration-dependent manner, by the photoaffinity probe 8-N3-GDP[32P]-d-mannose. The purified enzyme was stable for several months when kept in the frozen state. The 37-kDa band was subjected to protein sequencing and one peptide sequence of 25 amino acids showed over 80% identity to GDP-mannose pyrophosphorylases of pig liver and Saccharomyces cerevesiae. In contrast to some other bacterial GDP-mannose pyrophosphorylases, the mycobacterial enzyme was not multifunctional and did not have phosphomannose isomerase or phosphoglucose isomerase activity. Also, in contrast to the pig liver enzyme which uses mannose-1-P or glucose-1-P plus GTP to synthesize either GDP-mannose or GDP-glucose, the mycobacterial enzyme was specific for mannose-1-P as the sugar phosphate substrate. The enzyme was also relatively specific for GTP as the nucleoside triphosphate substrate. ITP was about 18% as effective as GTP, but ATP, CTP, and UTP were inactive. The activity of the enzyme was inhibited by GDP-glucose and glucose-1-P, although neither was a substrate for this enzyme. The pH optimum for the enzyme was 8.0, and Mg2+ was the best cation with optimum activity at about 5 mM. This enzyme is important for producing the activated form of mannose for formation of cell wall lipoarabinomannan and various mannose-containing glycolipids and polysaccharides.

Topics: Amino Acid Sequence; Animals; Enzyme Stability; Glucosephosphates; Guanosine Diphosphate Mannose; Guanosine Diphosphate Sugars; Guanosine Triphosphate; Hydrogen-Ion Concentration; Inosine Triphosphate; Kinetics; Magnesium; Mannose-6-Phosphate Isomerase; Mannosephosphates; Molecular Sequence Data; Molecular Weight; Mycobacterium smegmatis; Nucleotidyltransferases; Photoaffinity Labels; Sequence Analysis; Sequence Homology, Amino Acid; Substrate Specificity

In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] mannose into mannose phosphates, GDP-mannose, GDP-fucose, dolichol-P-mannose, lipid-linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into mannose 1-phosphate, GDP-mannose, GDP-fucose, dolichol-P-mannose, and nascent glycoproteins. The incorporation of radioactivity into mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (

Topics: Carbohydrates; Cells, Cultured; Congenital Disorders of Glycosylation; Dolichol Monophosphate Mannose; Fibroblasts; Guanosine Diphosphate Fucose; Guanosine Diphosphate Mannose; Humans; Lipopolysaccharides; Mannosephosphates; Phosphotransferases (Phosphomutases)