Intrinsic lipid preferences and kinetic mechanism of Escherichia coli MurG
Intrinsic lipid preferences and kinetic mechanism of Escherichia coli MurG
复制标题
DOI：
10.1021/bi0256678
复制
发表时间：
2002-05-28
期刊：
影响因子：
2.9
通讯作者：
Walker, S
中科院分区：
文献类型：
--
作者：
Chen, L;Men, H;Walker, S
MUrG是参与肽聚糖合成胞内相的最后一种酶，是一种膜相关糖基转移酶，可将N-乙酰葡糖胺偶联至脂质连接的N-乙酰胞壁酸衍生物（脂质I）的C4羟基，形成β-连接二糖（脂质II），后者是肽聚糖的最小亚基。脂质I通过55个碳的十一异戊二烯基链锚定到细菌膜上。由于这种长的脂质链阻碍MurG的动力学分析，我们一直在研究含有缩短的脂质链的替代底物。现在，我们描述了内在的脂质偏好的MurG和显示，最佳的基板MurG在膜的情况下是不是天然基板。因此，虽然十一异戊二烯基载体脂质可能对肽聚糖生物合成途径中的某些步骤至关重要，但MurG并不需要-事实上，也不优选。使用合成的底物类似物和含有不同长度的脂质链的产品，以及合成的死端受体类似物，我们还表明，MurG遵循一个强制性的有序Bi Bi机制，其中供体糖首先结合。该信息应有助于获得与底物结合的MurG晶体。这是一个重要的目标，因为MurG属于NDP-糖基转移酶的主要超家族，对于该超家族，还没有解决含有完整底物的结构。
MUrG, the last enzyme involved in the intracellular phase of peptidoglycan synthesis, is a membrane-associated glycosyltransferase that couples N-acetyl glucosamine to the C4 hydroxyl of a lipid-linked N-acetyl muramic acid derivative (lipid I) to form the beta-linked disaccharide (lipid II) that is the minimal subunit of peptidoglycan. Lipid I is anchored to the bacterial membrane by a 55 carbon undecaprenyl chain. Because this long lipid chain impedes kinetic analysis of MurG, we have been investigating alternative substrates containing shortened lipid chains. We now describe the intrinsic lipid preferences of MurG and show that the optimal substrate for MurG in the absence of membranes is not the natural substrate. Thus, while the undecaprenyl carrier lipid may be critical for certain steps in the biosynthetic pathway to peptidoglycan, it is not required-in fact, is not preferred-by MurG. Using synthetic substrate analooues and products containing different length lipid chains, as well as a synthetic dead-end acceptor analogue, we have also shown that MurG follows a compulsory ordered Bi Bi mechanism in which the donor sugar binds first. This information should facilitate obtaining, crystals of MurG with substrates bound. an important goal because MurG belongs to a major superfamily of NDP-glycosyltransferases for which no structures containing intact substrates have yet been solved.