Holst O, Bock K, Brade L, Brade H
Division of Biochemical Microbiology, Forschungsinstitut Borstel, Germany.
Eur J Biochem. 1995 Apr 1;229(1):194-200.
The lipopolysaccharide from the recombinant strain Escherichia coli F515-140 containing the cloned gene gseA [3-deoxy-D-manno-octulopyranosonic acid (Kdo) transferase] from Chlamydia psittaci 6BC was isolated and sequentially de-O-acylated and de-N-acylated. The products were separated by high-performance anion-exchange chromatography into three fractions, two of which contained a single compound. Their structures were elucidated by high-field NMR spectroscopy as alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P2 (compound 1) (tetrasaccharide bisphosphate) [Holst, O., Broer, W., Thomas-Oates, J. E., Mamat, U. & Brade, H. (1993) Eur. J. Biochem. 214, 703-710] and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)-]-alpha-Kdo-(2-->4)-alpha-Kdo- (2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P2 (compound 4) (hexasaccharide bisphosphate). The third fraction comprised two pentasaccharide bisphosphates, which could be separated by affinity chromatography using an immobilized monoclonal antibody specific for the trisaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. The bound fraction was identified as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D- GlcN-(1-->6)-alpha-D-GlcN 1,4'-P2 (compound 2) [Holst, O., Broer, W., Thomas-Oates, J. E., Mamat, U. & Brade, H. (1993) Eur. J. Biochem. 214, 703-710], whereas the unbound fraction was identified as alpha-Kdo-(2-->4)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6 )- alpha-D-GlcN 1,4'-P2 (compound 3). This novel Kdo tetrasaccharide extends our knowledge on multifunctional Kdo transferases.
从含有鹦鹉热衣原体6BC克隆基因gseA [3-脱氧-D-甘露-辛吡喃糖酸（Kdo）转移酶]的重组大肠杆菌F515-140中分离出脂多糖，并依次进行脱O-酰化和脱N-酰化。产物通过高效阴离子交换色谱法分离为三个馏分，其中两个馏分各含有一种单一化合物。通过高场核磁共振光谱法阐明了它们的结构，分别为α-Kdo-(2→4)-α-Kdo-(2→6)-β-D-GlcN-(1→6)-α-D-GlcN 1,4'-P2（化合物1）（四糖二磷酸）[霍尔斯特，O.，布勒，W.，托马斯-奥茨，J. E.，马马特，U. & 布拉德，H.（1993年）《欧洲生物化学杂志》214卷，703 - 710页]和α-Kdo-(2→4)-[α-Kdo-(2→8)-]-α-Kdo-(2→4)-α-Kdo-(2→6)-β-D-GlcN-(1→6)-α-D-GlcN 1,4'-P2（化合物4）（六糖二磷酸）。第三个馏分包含两种五糖二磷酸，它们可以通过使用对三糖α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo具有特异性的固定化单克隆抗体进行亲和色谱法分离。结合的馏分被鉴定为α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo-(2→6)-β-D-GlcN-(1→6)-α-D-GlcN 1,4'-P2（化合物2）[霍尔斯特，O.，布勒，W.，托马斯-奥茨，J. E.，马马特，U. & 布拉德，H.（1993年）《欧洲生物化学杂志》214卷，703 - 710页]，而未结合的馏分被鉴定为α-Kdo-(2→4)-α-Kdo-(2→4)-α-Kdo-(2→6)-β-D-GlcN-(1→6)-α-D-GlcN 1,4'-P2（化合物3）。这种新型的Kdo四糖扩展了我们对多功能Kdo转移酶的认识。
