Manat Guillaume, El Ghachi Meriem, Auger Rodolphe, Baouche Karima, Olatunji Samir, Kerff Frédéric, Touzé Thierry, Mengin-Lecreulx Dominique, Bouhss Ahmed
Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris Sud, Bâtiment 430, F-91400, Orsay, France.
Centre d'Ingénierie des Protéines, Université de Liège, Institut de Physique B5a et Institut de Chimie B6a, Sart-Tilman, B-4000, Liège, Belgium.
PLoS One. 2015 Nov 11;10(11):e0142870. doi: 10.1371/journal.pone.0142870. eCollection 2015.
Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C55-PP molecules on the same (outer) side of the plasma membrane.
先前在大肠杆菌中鉴定出几种具有十一异戊二烯基焦磷酸（C55-PP）磷酸酶活性的整合膜蛋白，它们属于两个不同的蛋白家族：BacA蛋白，其占大肠杆菌细胞膜中检测到的C55-PP磷酸酶活性的75%，以及PAP2磷脂酸磷酸酶家族的三个成员，即PgpB、YbjG和LpxT。这一步去磷酸化是为了提供C55-P载体脂质，其在各种细胞壁聚合物的生物合成中起核心作用。我们在此报告对BacA蛋白的生化特性和膜拓扑结构的详细研究。确定了最佳活性条件，并且观察到该酶对C55-PP具有明显偏好的窄范围底物特异性。BacA蛋白序列比对揭示了两个特别保守的区域和几个不变残基，通过定点诱变方法以及互补的体外和体内活性测定对其在酶活性中的作用提出了质疑。鉴定出三个必需残基Glu21、Ser27和Arg174，这使我们能够提出该酶的催化机制。此处通过实验确定的BacA蛋白的膜拓扑结构并未验证先前基于程序预测的模型。它由七个跨膜区段组成，尤其包含两个带有高度保守活性位点残基的大的周质环。因此，我们的数据提供了证据，表明迄今为止鉴定出的所有不同的大肠杆菌C55-PP磷酸酶（BacA和PAP2）都在质膜的同一（外）侧催化C55-PP分子的去磷酸化。
