guanosine-triphosphate and mannose-1-phosphate

GDP-D-mannose pyrophosphorylase catalyzes the production of GDP-D-mannose, an intermediate product in the plant ascorbic acid (AsA) biosynthetic pathway. This enzyme is a key regulatory target in AsA biosynthesis and is encoded by VITAMIN C DEFECTIVE 1 (VTC1) in the Arabidopsis thaliana genome. Here, recombinant VTC1 was expressed, purified and crystallized. Diffraction data were obtained from VTC1 crystals grown in the absence and presence of substrate using X-rays. The ligand-free VTC1 crystal diffracted X-rays to 3.3 Å resolution and belonged to space group R32, with unit-cell parameters a = b = 183.6, c = 368.5 Å, α = β = 90, γ = 120°; the crystal of VTC1 in the presence of substrate diffracted X-rays to 1.75 Å resolution and belonged to space group P2

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Cloning, Molecular; Crystallization; Crystallography, X-Ray; Escherichia coli; Gene Expression; Guanosine Triphosphate; Isoenzymes; Mannosephosphates; Phosphoric Monoester Hydrolases; Plasmids; Recombinant Proteins; X-Ray Diffraction

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