Biosynthesis of nucleotide-activated D-glycero-D-manno-heptose

The glycan chain repeats of the S-layer glycoprotein of Aneurinibacillus th ermoaerophilus 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-activat ed form GDP-D-glycero-D-manno-heptose were cloned, sequenced, and overexpre ssed in Escherichia coli, and the corresponding enzymes GmhA, GmhB, GmhC, a nd GmhD were purified to homogeneity, The isomerase GmhA catalyzed the conv ersion of D-sedoheptulose 7-phosphate to D-glycero-D-manno-heptose 7-phosph ate, 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-cu-D-manno-heptose :L- phosphate was eventually activated with GTP by the pyrophosphorylase GmhD t o yield the final product GDP-D-glycero-alpha -D-manno-heptose. The interme diate and end products were analyzed by high performance liquid chromatogra phy. Nuclear magnetic resonance spectroscopy was used to confirm the struct ure of these substances. This is the first report of the biosynthesis of GD P-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.