title: Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.
abstract: 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.
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris Sud, Bâtiment 430, F-91400, Orsay, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Manat
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Institut de Physique B5a et Institut de Chimie B6a, Sart-Tilman, B-4000, Liège, Belgium.
authors.forename: Meriem
authors.initials: M
authors.lastname: El Ghachi
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris Sud, Bâtiment 430, F-91400, Orsay, France.
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Auger
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris Sud, Bâtiment 430, F-91400, Orsay, France.
authors.forename: Karima
authors.initials: K
authors.lastname: Baouche
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Institut de Physique B5a et Institut de Chimie B6a, Sart-Tilman, B-4000, Liège, Belgium.
authors.forename: Samir
authors.initials: S
authors.lastname: Olatunji
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Institut de Physique B5a et Institut de Chimie B6a, Sart-Tilman, B-4000, Liège, Belgium.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Kerff
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris Sud, Bâtiment 430, F-91400, Orsay, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Touzé
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris Sud, Bâtiment 430, F-91400, Orsay, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Mengin-Lecreulx
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), UMR 9198, CEA, CNRS, Université Paris Sud, Bâtiment 430, F-91400, Orsay, France.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Bouhss
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005816:Genetic Complementation Test; D005973:Glutamine / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010711:Phosphatidate Phosphatase / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D008055:Lipids; D008565:Membrane Proteins; C000608554:PgpB protein, E coli; C585154:YbjG protein, E coli; D005973:Glutamine; D012694:Serine; D001120:Arginine; C553096:LpxT protein, E coli; D017856:Phosphotransferases (Phosphate Group Acceptor); C488524:BacA protein, E coli; D010744:Phosphoric Monoester Hydrolases; D010711:Phosphatidate Phosphatase