Kneidinger B, Graninger M, Puchberger M, Kosma P, Messner P
Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, Gregor-Mendel-Strasse 33, A-1180 Wien, Austria.
J Biol Chem. 2001 Jun 15;276(24):20935-44. doi: 10.1074/jbc.M100378200. Epub 2001 Mar 28.
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.
嗜热栖热放线菌DSM 10155的S层糖蛋白的聚糖链重复序列含有D-甘油-D-甘露庚糖，该糖也被描述为革兰氏阴性菌脂多糖核心的组成成分。负责核苷酸活化形式GDP-D-甘油-D-甘露庚糖生物合成的四个基因被克隆、测序并在大肠杆菌中过表达，相应的酶GmhA、GmhB、GmhC和GmhD被纯化至同质。异构酶GmhA催化D-景天庚酮糖7-磷酸转化为D-甘油-D-甘露庚糖7-磷酸，磷酸激酶GmhB添加一个磷酸基团形成D-甘油-D-甘露庚糖1,7-二磷酸。磷酸酶GmhC去除C-7位的磷酸，中间产物D-甘油-α-D-甘露庚糖1-磷酸最终被焦磷酸化酶GmhD用GTP活化，生成最终产物GDP-D-甘油-α-D-甘露庚糖。中间产物和终产物通过高效液相色谱进行分析。核磁共振光谱用于确认这些物质的结构。这是革兰氏阳性菌中GDP-D-甘油-α-D-甘露庚糖生物合成的首次报道。此外，我们提出了一条L-甘油-D-甘露庚糖核苷酸活化形式的生物合成途径。
