Biosynthesis of nucleotide-activated D-glycero-D-manno-heptose.
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.
Kneidinger
B
B
Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, Gregor-Mendel-Strasse 33, A-1180 Wien, Austria.
Graninger
M
M
Puchberger
M
M
Kosma
P
P
Messner
P
P
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry; D001483:Base Sequence; D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005798:Genes, Bacterial; D006090:Gram-Negative Bacteria / Q000737:chemistry / Q000235:genetics; D006156:Guanosine Diphosphate Sugars / Q000096:biosynthesis* / Q000737:chemistry; D006539:Heptoses / Q000096:biosynthesis* / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D009876:Operon; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D017931:DNA Primers; D006156:Guanosine Diphosphate Sugars; D006539:Heptoses; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C055764:S-layer proteins; C026312:glycero-manno-heptose; C430874:GmhC protein, Aneurinibacillus thermoaerophilus; D010744:Phosphoric Monoester Hydrolases