PMID:11279237
| Citation |
Kneidinger, B, Graninger, M, Puchberger, M, Kosma, P and Messner, P (2001) Biosynthesis of nucleotide-activated D-glycero-D-manno-heptose. J. Biol. Chem. 276:20935-44 |
|---|---|
| Abstract |
The glycan chain repeats of the S-layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 contain d-glycero-d-manno-heptose, which has also been described as constituent of lipopolysaccharide cores of Gram-negative bacteria. The four genes required for biosynthesis of the nucleotide-activated form GDP-d-glycero-d-manno-heptose were cloned, sequenced, and overexpressed in Escherichia coli, and the corresponding enzymes GmhA, GmhB, GmhC, and GmhD were purified to homogeneity. The isomerase GmhA catalyzed the conversion of d-sedoheptulose 7-phosphate to d-glycero-d-manno-heptose 7-phosphate, and the phosphokinase GmhB added a phosphate group to form d-glycero-d-manno-heptose 1,7-bisphosphate. The phosphatase GmhC removed the phosphate in the C-7 position, and the intermediate d-glycero-alpha-d-manno-heptose 1-phosphate was eventually activated with GTP by the pyrophosphorylase GmhD to yield the final product GDP-d-glycero-alpha-d-manno-heptose. The intermediate and end products were analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to confirm the structure of these substances. This is the first report of the biosynthesis of GDP-d-glycero-alpha-d-manno-heptose in Gram-positive organisms. In addition, we propose a pathway for biosynthesis of the nucleotide-activated form of l-glycero-d-manno-heptose. |
| Links |
PubMed Online version:10.1074/jbc.M100378200 |
| Keywords |
Amino Acid Sequence; Bacillaceae/chemistry; Bacillaceae/genetics; Bacillaceae/metabolism; Bacterial Proteins/biosynthesis; Bacterial Proteins/chemistry; Base Sequence; Carbohydrate Conformation; Chromatography, High Pressure Liquid; Cloning, Molecular; DNA Primers; Escherichia coli; Genes, Bacterial; Gram-Negative Bacteria/chemistry; Gram-Negative Bacteria/genetics; Guanosine Diphosphate Sugars/biosynthesis; Guanosine Diphosphate Sugars/chemistry; Heptoses/biosynthesis; Heptoses/chemistry; Membrane Glycoproteins/biosynthesis; Membrane Glycoproteins/chemistry; Molecular Sequence Data; Operon; Phosphoric Monoester Hydrolases/chemistry; Phosphoric Monoester Hydrolases/genetics; Phosphoric Monoester Hydrolases/metabolism; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Restriction Mapping; Sequence Alignment; Sequence Homology, Amino Acid |
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