A Leptospira interrogans enzyme with similarity to yeast Ste14p that methylates the 1-phosphate group of lipid A
A Leptospira interrogans enzyme with similarity to yeast Ste14p that methylates the 1-phosphate group of lipid A
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DOI：
10.1074/jbc.m506103200
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发表时间：
2005-08-26
影响因子：
4.8
通讯作者：
Raetz, CRH
中科院分区：
文献类型：
--
作者：
Hinckley, MB;Reynolds, CM;Raetz, CRH
与其他螺旋体不同，钩端螺旋体的细胞含有脂质A生物合成所需的所有大肠杆菌lpx基因的同源物，但它们合成一种修饰形式的脂多糖，据推测，这种脂多糖可以激活toll样受体2 (TLR2)，而不是TLR4。最近的测定揭示了一种前所未有的1-磷酸基团o -甲基化(Que-Gewirth, n.l.s., Ribeiro, a.a., Kalb, s.r., Cotter, r.j., Bulach, d.m., Adler， B., Saint Girons， I., Werts， C.和Raetz， c.r.h. (2004) J. Biol。化学，279,25420 - 25429)。负责选择性1-磷酸甲基化的酶活性以前没有被探索过。一种催化甲基从s -腺苷蛋氨酸（SAM）转移到大肠杆菌Kdo(2)-[4'- p -32]脂质A的1-磷酸部分的膜酶现已被发现。编码该酶的基因是基于磷酸基甲基化在化学上类似于膜-水界面羧酸基甲基化的假设而确定的。数据库检索显示，L.疑问菌的候选基因（重命名为lmtA）与酵母异戊酰半胱氨酸羧基甲基转移酶具有远同源性，该基因由不育-14编码，可甲基化a型交配因子。在大肠杆菌（其脂质A通常缺乏1-磷酰乙基）或其他螺旋体中不存在lta的同源物。不合成脂质a lac背后的lmtA基因启动子的表达在低拷贝质粒导致的外观SAM-dependent甲基转移酶活性在大肠杆菌内膜和30%的甲基化内生大肠杆菌脂质a失活的ABC转运MsbA没有抑制甲基化新合成的脂质a .甲基化分析了大肠杆菌脂质谱和核磁共振谱证实phosphomethyl组的位置在哪里吗在1的位置。在人类细胞中，从表达lta的大肠杆菌中纯化的1-磷酸化甲基脂质A能有效激活TLR4，但不能激活TLR2。
Distinct from other spirochetes, cells of Leptospira interrogans contain orthologues of all the Escherichia coli lpx genes required for lipid A biosynthesis, but they synthesize a modified form of lipopolysaccharide that supposedly activates toll-like receptor 2 ( TLR2) instead of TLR4. The recent determination of the L. interrogans lipid A structure revealed an unprecedented O-methylation of its 1-phosphate group ( Que-Gewirth, N. L. S., Ribeiro, A. A., Kalb, S. R., Cotter, R. J., Bulach, D. M., Adler, B., Saint Girons, I., Werts, C., and Raetz, C. R. H. ( 2004) J. Biol. Chem. 279, 25420 - 25429). The enzymatic activity responsible for selective 1-phosphate methylation has not been previously explored. A membrane enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine ( SAM) to the 1-phosphate moiety of E. coli Kdo(2)-[4'-P-32] lipid A has now been discovered. The gene encoding this enzyme was identified based on the hypothesis that methylation of a phosphate group is chemically analogous to methylation of a carboxylate moiety at a membrane-water interface. Database searching revealed a candidate gene ( renamed lmtA) in L. interrogans showing distant homology to the yeast isoprenylcysteine carboxyl methyltransferase, encoded by sterile-14, which methylates the a-type mating factor. Orthologues of lmtA were not present in E. coli, the lipid A of which normally lacks the 1-phosphomethyl group, or in other spirochetes, which do not synthesize lipid A. Expression of the lmtA gene behind the lac promoter on a low copy plasmid resulted in the appearance of SAM-dependent methyltransferase activity in E. coli inner membranes and methylation of about 30% of the endogenous E. coli lipid A. Inactivation of the ABC transporter MsbA did not inhibit methylation of newly synthesized lipid A. Methylated E. coli lipid A was analyzed by mass spectrometry and NMR spectroscopy to confirm the location of the phosphomethyl group at the 1-position. In human cells, engineered to express the individual TLR subtypes, 1-phosphomethyl-lipid A purified from lmtA-expressing E. coli potently activated TLR4 but not TLR2.