A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum -: Purification, substrate specificity, and expression in Salmonella waaC mutants
A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum -: Purification, substrate specificity, and expression in Salmonella waaC mutants
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DOI：
10.1074/jbc.m301255200
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发表时间：
2003-05-02
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：
Kanipes, MI;Ribeiro, AA;Raetz, CRH
革兰氏阴性菌的脂多糖（LPS）核心结构域在外膜稳定性和宿主相互作用中起重要作用。对组装LPS核心的糖基转移酶的生物化学性质知之甚少。我们现在报告豆科根瘤菌甘露糖基转移酶LpcC的纯化和表征，该转移酶LpcC将甘露糖单元添加到LPS前体Kdo（2）-脂质IVA的内部3-脱氧-D-甘露-辛酮糖酸（Kdo）部分。使用GDP-甘露糖作为供体和Kdo（2）-[4 '-P-32]脂质IVA作为受体测定含有N-末端His标签的LpcC，并纯化至接近同质。N端测序证实纯化的酶是lpcC基因产物。体外产生的糖脂经弱酸水解后，甘露糖基化发生在Kdo（2）-[4 '-P-32]脂质IVA的Kdo内部残基上。LpcC需要含有两个Kdo部分的脂质受体底物，因为用脂质IVA或Kdo-脂质IVA未观察到活性。纯化的酶可以使用GDP-甘露糖或在较小程度上使用ADP-甘露糖（两者都具有α-异头构型）用于Kdo 2-[4 '-P-32]脂质IVA的糖基化。ADP-葡萄糖、UDP-葡萄糖、UDP-GlcNAc或UDP-半乳糖几乎没有活性或没有活性。鼠伤寒沙门氏菌waaC突变体缺乏整合LPS内部L-甘油-D-甘露庚糖部分的酶，当与lpcC互补时，可以用O-抗原重新获得LPS。大肠杆菌无庚糖waaC-waaF缺失突变体表达R.豆科植物lpcC基因同样产生由Kdo（2）-脂质A加上单个甘露糖残基组成的杂合LPS种类。我们的研究结果表明，异源lpcC表达可用于修饰沙门氏菌和大肠杆菌的结构。coli LPS核心。
The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyI transferase LpcC, which adds a mannose unit to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo(2)-lipid IVA. LpcC containing an N-terminal His, tag was assayed using GDP-mannose as the donor and Kdo(2)-[4'-P-32]lipid IVA as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue Of Kdo(2)-[4'-P-32]lipid IVA. A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IVA or Kdo-lipid IVA. The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the alpha-anomeric configuration) for the glycosylation of Kdo2-[4'-P-32]lipid IVA. Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner L-glycero-D-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo(2)-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells.