Active site mapping of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, catalyzing the first membrane step of peptidoglycan biosynthesis
Active site mapping of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, catalyzing the first membrane step of peptidoglycan biosynthesis
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DOI：
10.1021/bi8006274
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发表时间：
2008-08-26
期刊：
影响因子：
2.9
通讯作者：
Bouhss, Ahmed
中科院分区：
文献类型：
--
作者：
Al-Dabbagh, Bayan;Henry, Xavier;Bouhss, Ahmed
MraY转移酶是一种整合的膜蛋白，其催化肽聚糖生物合成的重要步骤，即磷酸-N-乙酰胞壁酰-五肽基序转移到十一异戊二烯磷酸载体脂质上。它属于真核和原核异戊二烯基糖转移酶的大超家族。目前还没有关于这个超家族的任何成员的3D结构的报道，而迄今为止，MraY是唯一一个被成功纯化到同质的蛋白质。19个极性残基位于MraY的5个细胞质片段中，在MraY直向同源物的序列中作为不变量出现。这些不变的残基中有一定数量的残基在整个超家族中是保守的。为了评估这些残基在催化过程中的重要性，使用枯草芽孢杆菌MraY作为模型进行定点诱变。14个残基被证明是必要的MraY活性的体内功能互补试验，使用构建的条件mraY突变株。相应的突变蛋白进行纯化和生化特性。这些突变都没有显着影响的核苷酸和磷酸底物的结合，但k（猫），，在几乎所有的情况下显着降低。因此，活性的重要残基似乎分布在所有的细胞质片段中，表明这五个区域有助于催化位点的结构。我们的数据表明，D98残基是不变的，在整个超家族应该参与的脂质底物的去质子化过程中的催化过程。
The MraY transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. No 3D structure has been reported for any member of this superfamily, and to date MraY is the only protein that has been successfully purified to homogeneity. Nineteen polar residues located in the five cytoplasmic segments of MraY appeared as invariants in the sequences of MraY orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the Bacillus subtilis MraY as a model. Fourteen residues were shown to be essential for MraY activity by an in vivo functional complementation assay using a constructed conditional mraY mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of the nucleotidic and lipidic substrates, but the k(cat),, was dramatically reduced in almost all cases. The important residues for activity therefore appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the D98 residue that is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process.