Gronow S, Brabetz W, Brade H
Division of Medical and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany.
Eur J Biochem. 2000 Nov;267(22):6602-11. doi: 10.1046/j.1432-1327.2000.01754.x.
Heptosyltransferase II, encoded by the waaF gene of Escherichia coli, is a glycosyltransferase involved in the synthesis of the inner core region of lipopolysaccharide. The gene was subcloned from plasmid pWSB33 [Brabetz, W., Müller-Loennies, S., Holst, O. & Brade, H. (1997) Eur. J. Biochem. 247, 716-724] into a shuttle vector for the expression in the gram-positive host Corynebacterium glutamicum. The in vitro activity of the enzyme was investigated in comparison to that of heptosyltransferase I (WaaC) using as a source for the sugar nucleotide donor, ADP-LglyceroDmanno-heptose, a low molecular mass filtrate from a DeltawaaCF E. coli strain. Synthetic lipid A analogues varying in the acylation or phosphorylation pattern or both were tested as acceptors for the subsequent transfer of 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) and heptose by successive action of Kdo transferase (WaaA), heptosyltransferase I (WaaC) and heptosyltransferase II (WaaF). The reaction products were characterized after separation by TLC and blotting with monoclonal antibodies specific for the acceptor, the intermediates and the final products.
庚糖基转移酶II由大肠杆菌的waaF基因编码，是一种参与脂多糖内核区域合成的糖基转移酶。该基因从质粒pWSB33 [布拉贝茨，W.，米勒 - 伦尼斯，S.，霍尔斯特，O. & 布拉德，H.（1997年）《欧洲生物化学杂志》247，716 - 724]亚克隆到穿梭载体中，以便在革兰氏阳性宿主谷氨酸棒杆菌中表达。与庚糖基转移酶I（WaaC）相比，使用来自ΔwaaCF大肠杆菌菌株的低分子量滤液ADP-L-甘油-D-甘露庚糖作为糖核苷酸供体来源，研究了该酶的体外活性。测试了酰化或磷酸化模式或两者都不同的合成类脂A类似物作为后续通过Kdo转移酶（WaaA）、庚糖基转移酶I（WaaC）和庚糖基转移酶II（WaaF）的连续作用转移3-脱氧-D-甘露-辛-2-酮糖酸（Kdo）和庚糖的受体。通过薄层层析分离并用对受体、中间体和最终产物具有特异性的单克隆抗体进行印迹后，对反应产物进行了表征。
