Department of Biochemistry, Duke University Medical Center , Durham, North Carolina 27710, United States.
Biochemistry. 2014 Mar 4;53(8):1250-62. doi: 10.1021/bi4015665. Epub 2014 Feb 21.
There are five distinct core structures in the lipopolysaccharides of Escherichia coli and at least two in Salmonella isolates, which vary principally in the outer core oligosaccharide. Six outer core glycosyltransferases, E. coli K-12 WaaG, WaaB, and WaaO and Salmonella typhimurium WaaI, WaaJ, and WaaK, were cloned, overexpressed, and purified. A novel substrate for WaaG was isolated from ΔwaaG E. coli overexpressing the lipid A phosphatase lpxE and the lipid A late acyltransferase lpxM. The action of lpxE and lpxM in the ΔwaaG background yielded heptose2-1-dephospho Kdo2-lipid A, a 1-dephosphorylated hexa-acylated lipid A with the inner core sugars that is easily isolated by organic extraction. Using this structurally defined acceptor and commercially available sugar nucleotides, each outer core glycosyltransferases was assayed in vitro. We show that WaaG and WaaB add a glucose and galactose sequentially to heptose2-1-dephospho Kdo2-lipid A. E. coli K-12 WaaO and S. typhimurium WaaI add a galactose to the WaaG/WaaB product but can also add a galactose to the WaaG product directly without the branched core sugar added by WaaB. Both WaaI and WaaO require divalent metal ions for optimal activity; however, WaaO, unlike WaaI, can add several glucose residues to its lipid acceptor. Using the product of WaaG, WaaB, and WaaI, we show that S. typhimurium WaaJ and WaaK transfer a glucose and N-acetylglucosamine, respectively, to yield the full outer core. This is the first demonstration of the in vitro assembly of the outer core of the lipopolysaccharide using defined lipid A-oligosaccharide acceptors and sugar donors.
大肠杆菌脂多糖中有五个不同的核心结构，沙门氏菌分离株中至少有两个，其主要区别在外核心寡糖。克隆、过表达和纯化了六种外核心糖基转移酶，大肠杆菌 K-12 WaaG、WaaB 和 WaaO 以及鼠伤寒沙门氏菌 WaaI、WaaJ 和 WaaK。从过表达脂磷壁酸磷酸酶 lpxE 和脂磷壁酸晚期酰基转移酶 lpxM 的Δ waaG 大肠杆菌中分离出 WaaG 的一种新底物。在Δ waaG 背景下，lpxE 和 lpxM 的作用产生了七糖 2-1-去磷酸化 Kdo2-脂磷壁酸，这是一种 1-去磷酸化的六酰化脂磷壁酸，带有内核心糖，很容易通过有机提取分离。使用这种结构定义的受体和商业可得的糖核苷酸，体外测定了每种外核心糖基转移酶。我们表明，WaaG 和 WaaB 依次向七糖 2-1-去磷酸化 Kdo2-脂磷壁酸添加葡萄糖和半乳糖。大肠杆菌 K-12 WaaO 和鼠伤寒沙门氏菌 WaaI 向 WaaG/WaaB 产物添加半乳糖，但也可以直接向 WaaG 产物添加半乳糖，而无需 WaaB 添加支化核心糖。WaaI 和 WaaO 都需要二价金属离子才能发挥最佳活性；然而，与 WaaI 不同，WaaO 可以将几个葡萄糖残基添加到其脂质受体上。使用 WaaG、WaaB 和 WaaI 的产物，我们表明鼠伤寒沙门氏菌 WaaJ 和 WaaK 分别转移葡萄糖和 N-乙酰葡萄糖胺，以生成完整的外核心。这是首次使用定义的脂磷壁酸-寡糖受体和糖供体在体外组装脂多糖外核心的证明。
