Stead Christopher, Tran An, Ferguson Donald, McGrath Sara, Cotter Robert, Trent Stephen
Department of Microbiology, J. H. Quillen College of Medicine, Johnson City, Tennessee 37614, USA.
J Bacteriol. 2005 May;187(10):3374-83. doi: 10.1128/JB.187.10.3374-3383.2005.
The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IV(A). In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.
脂多糖（LPS）的脂质A结构域将其锚定在外膜上，通常是一种被酰化和磷酸化的葡糖胺二糖。核心碳水化合物结构域和O抗原碳水化合物结构域通过八碳糖3-脱氧-D-甘露糖辛酮酸（称为Kdo）与脂质A部分相连。幽门螺杆菌LPS的特征是脂质A上连接有单个Kdo残基，由此推测其体内存在单功能Kdo转移酶（WaaA）。然而，我们使用体外检测系统证明，幽门螺杆菌WaaA是一种双功能酶，可将两个Kdo糖转移至四酰化脂质A前体脂质IV（A）。在本研究中，我们报告了在幽门螺杆菌膜中发现一种Kdo水解酶，它能够从Kdo2-脂质A上去除外部的Kdo糖。Kdo基团的酶促去除依赖于脂质A结构域上1-磷酸基团的预先去除，对反应产物的质谱分析证实了Kdo修剪酶对单个Kdo残基的酶促去除。这是对参与革兰氏阴性菌LPS修饰的Kdo水解酶的首次表征。
