Accumulation of the lipid A precursor UDP-2,3-diacylglucosamine in an Escherichia coli mutant lacking the lpxH gene.
Accumulation of the lipid A precursor UDP-2,3-diacylglucosamine in an Escherichia coli mutant lacking the lpxH gene.
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DOI：
10.1074/jbc.m204068200
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发表时间：
2002-07-19
影响因子：
4.8
通讯作者：
Raetz, CRH
中科院分区：
文献类型：
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作者：
Babinski, KJ;Kanjilal, SJ;Raetz, CRH
lpxH基因编码UDP-2，3-二酰基葡糖胺特异性焦磷酸酶，其催化大肠杆菌中脂质A生物合成的第四步。为了证实lpxH的功能，我们构建了KB 321/pKJB 5。该菌株在lpxH的染色体拷贝中含有卡那霉素插入元件，由质粒pKJB 5补充，质粒pKJB 5对复制具有温度敏感性并携带lpxH（+）。KB 21/pKJB 5在30 ℃生长，但在44 ℃失去活力，表明lpxH是必需的。CDP-甘油二酯水解酶（Cdh）在体外催化与LpxH相同的反应，但不是必需的，不能弥补LpxH的缺失。细胞提取物中Cdh的存在干扰LpxH测定。因此，我们构建了KB 25/pKJB 5，其包含cdh的框内缺失和lpxH中的卡那霉素插入突变，被pKJB 5覆盖。当KB 25/pKJB 5细胞在44 ℃下生长时，存活力丧失，并且所有体外LpxH活性被消除。在44 ℃失去覆盖质粒后，与合成UDP-2，3-二酰基葡糖胺一起迁移的脂质在KB 25/pKJB 5中积累。通过LpxH将该物质转化为预期产物2，3-二酰基葡糖胺1-磷酸盐和UMP。铜绿假单胞菌含有两个与大肠杆菌具有序列相似性的蛋白质。coli LpxH.更同源的蛋白质在体外催化UDP-2，3-二酰基葡糖胺水解。相应的基因在44 ℃下与YB 25/pKJB 5互补，但同源性较低的基因不互补。UDP-2，3-二酰基葡糖胺在我们的lpxH突变体中的积累与脂质A二糖合酶LpxB（途径中的下一个酶）不能缩合两个UDP-2，3-二酰基葡糖胺分子，而是利用UDP-2，3-二酰基葡糖胺作为其供体和2，3-二酰基葡糖胺1-磷酸作为其受体的观察结果一致。
The lpxH gene encodes the UDP-2,3-diacylglucosamine-specific pyrophosphatase that catalyzes the fourth step of lipid A biosynthesis in Escherichia coli. To confirm the function of lpxH, we constructed KB321/pKJB5. This strain contains a kanamycin insertion element in the chromosomal copy of lpxH, complemented by plasmid pKJB5, which is temperature-sensitive for replication and harbors lpxH(+). KB21/pKJB5 grows at 30 degreesC but loses viability at 44 degreesC, demonstrating that lpxH is essential. CDP-diglyceride hydrolase (Cdh) catalyzes the same reaction as LpxH in vitro but is nonessential and cannot compensate for the absence of LpxH. The presence of Cdh in cell extracts interferes with the LpxH assay. We therefore constructed KB25/ pKJB5, which contains both an in-frame deletion of cdh and a kanamycin insertion mutation in lpxH, covered by pKJB5. When KB25/pKJB5 cells are grown at 44 degreesC, viability is lost, and all in vitro LpxH activity is eliminated. A lipid migrating with synthetic UDP-2,3-diacylglucosamine accumulates in KB25/pKJB5 following loss of the covering plasmid at 44 degreesC. This material was converted to the expected products, 2,3-diacylglucosamine 1-phosphate and UMP, by LpxH. Pseudomonas aeruginosa contains two proteins with sequence similarity to E. coli LpxH. The more homologous protein catalyzes UDP-2,3-diacylglucosamine hydrolysis in vitro. The corresponding gene complements YB25/pKJB5 at 44 degreesC, but the less homologous gene does not. The accumulation of UDP-2,3-diacylglucosamine in our lpxH mutant is consistent with the observation that the lipid A disaccharide synthase LpxB, the next enzyme in the pathway, cannot condense two UDP-2,3-diacylglucosamine molecules, but instead utilizes UDP-2,3-diacylglucosamine as its donor and 2,3-diacylglucosamine 1-phosphate as its acceptor.