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Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis

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Abstract

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.

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Acknowledgements

This work was supported by the Belgian program of Interuniversity Attraction Poles initiated by the Federal Office for Scientific Technical and Cultural Affairs (IAP no. P7/44), the FRS-FNRS (MIS F.4518.12, IISN 4.4503.11), the Tournesol/Hubert Curien partnership between Belgium and France (R.CFRA.1567), the Science Foundation Ireland (Grant Number 12/IA/1255), the Agence Nationale de la Recherche (Bactoprenyl project, ANR-11-BSV3-002), the Centre National de la Recherche Scientifique and the University of Paris-Sud (UMR 9198) and the Aix-Marseille University. The assistance and support of beamline scientists at the Advanced Photon Source (23-ID) are acknowledged.

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Correspondence to Thierry Touzé, Martin Caffrey or Frédéric Kerff.

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Meriem El Ghachi, Nicole Howe, Rodolphe Auger and Alexandre Lambion contributed equally.

Data deposition: coordinates of the bsPgpB crystal structure have been deposited in the Protein Data Bank (PDB id code 5JKI).

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Ghachi, M.E., Howe, N., Auger, R. et al. Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis . Cell. Mol. Life Sci. 74, 2319–2332 (2017). https://doi.org/10.1007/s00018-017-2464-6

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