Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis.
Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.
Centre d'Ingénierie des Protéines, InBioS, Université de Liège, allée du 6 Août 19, Bât B5a, 4000, Liège, Belgium.
Meriem El
ME
Ghachi
Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.
Nicole
N
Howe
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
Rodolphe
R
Auger
Centre d'Ingénierie des Protéines, InBioS, Université de Liège, allée du 6 Août 19, Bât B5a, 4000, Liège, Belgium.
Alexandre
A
Lambion
Laboratoire de Chimie Bactérienne UMR 7283, Aix-Marseille Université, Marseille, France.
Annick
A
Guiseppi
Centre d'Ingénierie des Protéines, InBioS, Université de Liège, allée du 6 Août 19, Bât B5a, 4000, Liège, Belgium.
François
F
Delbrassine
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
Guillaume
G
Manat
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
Sophie
S
Roure
Laboratoire de Chimie Bactérienne UMR 7283, Aix-Marseille Université, Marseille, France.
Sabine
S
Peslier
Centre d'Ingénierie des Protéines, InBioS, Université de Liège, allée du 6 Août 19, Bât B5a, 4000, Liège, Belgium.
Eric
E
Sauvage
Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.
Lutz
L
Vogeley
Centre d'Ingénierie des Protéines, InBioS, Université de Liège, allée du 6 Août 19, Bât B5a, 4000, Liège, Belgium.
Juan-Carlos
JC
Rengifo-Gonzalez
Centre d'Ingénierie des Protéines, InBioS, Université de Liège, allée du 6 Août 19, Bât B5a, 4000, Liège, Belgium.
Paulette
P
Charlier
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
Dominique
D
Mengin-Lecreulx
Laboratoire de Chimie Bactérienne UMR 7283, Aix-Marseille Université, Marseille, France.
Maryline
M
Foglino
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France. thierry.touze@i2bc.paris-saclay.fr.
Thierry
T
Touzé
Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland. martin.caffrey@tcd.ie.
Martin
M
Caffrey
Centre d'Ingénierie des Protéines, InBioS, Université de Liège, allée du 6 Août 19, Bât B5a, 4000, Liège, Belgium. fkerff@ulg.ac.be.
Frédéric
F
Kerff
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010711:Phosphatidate Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010715:Phosphatidylglycerols / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity
D010715:Phosphatidylglycerols; C034358:phosphatidylglycerophosphate; C406242:phosphatidic acid phosphatase type 2; D010711:Phosphatidate Phosphatase
Bacterial lipids metabolism; Membrane protein structure; Peptidoglycan-related lipid; Undecaprenyl phosphate