Oxygen requirement for the biosynthesis of the S-2-hydroxymyristate moiety in Salmonella typhimurium lipid A -: Function of LpxO, a new Fe2+/α-ketoglutarate-dependent dioxygenase homologue
Oxygen requirement for the biosynthesis of the S-2-hydroxymyristate moiety in Salmonella typhimurium lipid A -: Function of LpxO, a new Fe2+/α-ketoglutarate-dependent dioxygenase homologue
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DOI：
10.1074/jbc.m005779200
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发表时间：
2000-10-20
影响因子：
4.8
通讯作者：
Raetz, CRH
中科院分区：
文献类型：
--
作者：
Gibbons, HS;Lin, S;Raetz, CRH
某些革兰氏阴性菌（包括鼠伤寒沙门氏菌和铜绿假单胞菌）的脂质A分子可能含有二级S-2-羟酰基链。S.鼠伤寒沙门氏菌最近被证明以PhoP/PhoQ依赖的方式合成其S-2-羟基肉豆蔻酸酯修饰的脂质A，这表明2-OH基团在发病机制中可能起作用。从S.在存在或不存在O-2的情况下生长的鼠伤寒沙门氏菌。在厌氧条件下，没有形成含2-羟基肉豆蔻酸酯的脂质A。在O-18（2）存在下生长的细胞中，脂质A的基质辅助激光解吸/电离飞行时间质谱证实分子氧直接掺入2-羟基酰基修饰的脂质A中。使用几种充分表征的双加氧酶蛋白质序列作为探针，tBLASTn搜索揭示了未分配的开放阅读框，其与已知产生2-羟基酰化脂质A分子的细菌中的哺乳动物胆甾醇/天冬酰胺酰β-羟化酶相似。色葡萄将鼠伤寒沙门氏菌乙酰化/天冬酰胺酰β-羟化酶同源物（命名为lpxO）克隆到pBluescriptSK中，并在不含lpxO的大肠杆菌K-12中表达。对所得构建体的分析显示，lpxO表达足以诱导大肠杆菌中2-羟基肉豆蔻酸酯修饰的脂质A的O-2依赖性形成。杆菌LpxO很可能是一种新的Fe 2 +/α-酮戊二酸依赖性双加氧酶，催化脂质A（或关键前体）的羟基化。色葡萄鼠伤寒lpxO基因编码302个氨基酸的多肽，在两端具有预测的膜锚定序列。我们推测，2-羟基肉豆蔻酸链内感染的巨噬细胞内的脂多糖释放可能会被转换为2-羟基肉豆蔻酰辅酶A，一个很好的特点，蛋白N-肉豆蔻酰转移酶的有效抑制剂。
Lipid A molecules of certain Gram-negative bacteria, including Salmonella typhimurium and Pseudomonas aeruginosa, may contain secondary S-2-hydroxyacyl chains. S. typhimurium has recently been shown to synthesize its S-2-hydroxymyristate-modified lipid A in a PhoP/PhoQ-dependent manner, suggesting a possible role for the 2-OH group in pathogenesis, We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Lipid A was extracted from a PhoP-constitutive mutant of S. typhimurium grown in the presence or absence of O-2. Under anaerobic conditions, no 2-hydroxymyristate-containing lipid A was formed. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipid A from cells grown in the presence of O-18(2) confirmed the direct incorporation of molecular oxygen into 2-hydroxyacyl-modified lipid A. Using several well characterized dioxygenase protein sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl/asparaginyl beta -hydroxylases in bacteria known to make 2-hydroxyacylated lipid A molecules. The S. typhimurium aspartyl/asparaginyl beta -hydroxylase homologue (designated lpxO) was cloned into pBluescriptSK and expressed in Escherichia coli K-12, which does not contain lpxO. Analysis of the resulting construct revealed that lpxO expression is sufficient to induce O-2-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO very likely is a novel Fe2+/alpha -ketoglutarate-dependent dioxygenase that catalyzes the hydroxylation of lipid A (or of a key precursor). The S. typhimurium lpxO gene encodes a polypeptide of 302 amino acids with predicted membrane-anchoring sequences at both ends. We hypothesize that 2-hydroxymyristate chains released from lipopolysaccharide inside infected macrophages might be converted to 2-hydroxymyristoyl coenzyme A, a well characterized, potent inhibitor of protein N-myristoyl transferase.