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

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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References

  1. 1.

    Sigal YJ, McDermott MI, Morris AJ (2005) Integral membrane lipid phosphatases/phosphotransferases: common structure and diverse functions. Biochem J 387:281–293. doi:10.1042/BJ20041771

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Manat G, Roure S, Auger R et al (2014) Deciphering the metabolism of undecaprenyl-phosphate: the bacterial cell-wall unit carrier at the membrane frontier. Microb Drug Resist 20:199–214. doi:10.1089/mdr.2014.0035

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Lei KJ, Shelly LL, Pan CJ et al (1993) Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a. Science 262:580–583

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Cullen TW, Giles DK, Wolf LN et al (2011) Helicobacter pylori versus the host: remodeling of the bacterial outer membrane is required for survival in the gastric mucosa. PLoS Pathog 7:e1002454. doi:10.1371/journal.ppat.1002454

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Fournier J-B, Rebuffet E, Delage L et al (2014) The Vanadium Iodoperoxidase from the marine flavobacteriaceae species Zobellia galactanivorans reveals novel molecular and evolutionary features of halide specificity in the vanadium haloperoxidase enzyme family. Appl Environ Microbiol 80:7561–7573. doi:10.1128/AEM.02430-14

    Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Comba S, Menendez-Bravo S, Arabolaza A, Gramajo H (2013) Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor. Microb Cell Fact 12:9. doi:10.1186/1475-2859-12-9

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Touzé T, Tran AX, Hankins JV et al (2008) Periplasmic phosphorylation of lipid A is linked to the synthesis of undecaprenyl phosphate. Mol Microbiol 67:264–277. doi:10.1111/j.1365-2958.2007.06044.x

    Article  PubMed  Google Scholar 

  8. 8.

    Chae M, Carman GM (2013) Characterization of the yeast actin patch protein app1p phosphatidate phosphatase. J Biol Chem 288:6427–6437. doi:10.1074/jbc.M112.449629

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Peschel A (2002) How do bacteria resist human antimicrobial peptides? Trends Microbiol 10:179–186. doi:10.1016/S0966-842X(02)02333-8

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Nishi H, Komatsuzawa H, Fujiwara T et al (2004) Reduced content of lysyl-phosphatidylglycerol in the cytoplasmic membrane affects susceptibility to moenomycin, as well as vancomycin, gentamicin, and antimicrobial peptides, in Staphylococcus aureus. Antimicrob Agents Chemother 48:4800–4807. doi:10.1128/AAC.48.12.4800-4807.2004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Kuhn S, Slavetinsky CJ, Peschel A (2015) Synthesis and function of phospholipids in Staphylococcus aureus. Int J Med Microbiol 305:196–202. doi:10.1016/j.ijmm.2014.12.016

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Raetz CR (1986) Molecular genetics of membrane phospholipid synthesis. Annu Rev Genet 20:253–295. doi:10.1146/annurev.ge.20.120186.001345

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Ratledge C, Wilkinson SG (1988) Microbial lipids vol. 1. Academic Presss, London

    Google Scholar 

  14. 14.

    Goldberg DE, Rumley MK, Kennedy EP (1981) Biosynthesis of membrane-derived oligosaccharides: a periplasmic phosphoglyceroltransferase. Proc Natl Acad Sci USA 78:5513–5517

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Koch HU, Haas R, Fischer W (1984) The role of lipoteichoic acid biosynthesis in membrane lipid metabolism of growing Staphylococcus aureus. Eur J Biochem 138:357–363

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Icho T, Raetz CRH (1983) Multiple genes for membrane-bound phosphatases in Escherichia coli and their action on phospholipid precursors. J Bacteriol 153:722–730

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Lu Y-H, Guan Z, Zhao J, Raetz CRH (2011) Three phosphatidylglycerol-phosphate phosphatases in the inner membrane of Escherichia coli. J Biol Chem 286:5506–5518. doi:10.1074/jbc.M110.199265

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Cantagrel V, Lefeber DJ (2011) From glycosylation disorders to dolichol biosynthesis defects: a new class of metabolic diseases. J Inherit Metab Dis 34:859–867. doi:10.1007/s10545-011-9301-0

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Apfel CM, Takács B, Fountoulakis M et al (1999) Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene. J Bacteriol 181:483–492

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Siewert G, SJ (1967) Bacitracin: an inhibitor of the dephosphorylation of lipid pyrophosphate, an intermediate in the biosynthesis of the peptidoglycan of bacterial cell wall. Proc Natl Acad Sci USA 57:767–773

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    El Ghachi M, Bouhss A, Blanot D, Mengin-Lecreulx D (2004) The bacA gene of Escherichia coli encodes an undecaprenyl pyrophosphate phosphatase activity. J Biol Chem 279:30106–30113. doi:10.1074/jbc.M401701200

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    El Ghachi M, Derbise A, Bouhss A, Mengin-Lecreulx D (2005) Identification of multiple genes encoding membrane proteins with undecaprenyl pyrophosphate phosphatase (UppP) activity in Escherichia coli. J Biol Chem 280:18689–18695. doi:10.1074/jbc.M412277200

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Bernard R, El Ghachi M, Mengin-Lecreulx D et al (2005) BcrC from Bacillus subtilis acts as an undecaprenyl pyrophosphate phosphatase in bacitracin resistance. J Biol Chem 280:28852–28857. doi:10.1074/jbc.M413750200

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Zhao H, Sun Y, Peters JM et al (2016) Depletion of undecaprenyl pyrophosphate phosphatases (UPP-Pases) disrupts cell envelope biogenesis in Bacillus subtilis. J Bacteriol. doi:10.1128/JB.00507-16

    Google Scholar 

  25. 25.

    Fan J, Jiang D, Zhao Y et al (2014) Crystal structure of lipid phosphatase Escherichia coli phosphatidylglycerophosphate phosphatase B. Proc Natl Acad Sci USA 111:7636–7640. doi:10.1073/pnas.1403097111

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Tong S, Lin Y, Lu S et al (2016) Structural insight into substrate selection and catalysis of lipid phosphate phosphatase PgpB in the cell membrane. J Biol Chem. doi:10.1074/jbc.M116.737874

    Google Scholar 

  27. 27.

    Isupov MN, Dalby AR, Brindley AA et al (2000) Crystal structure of dodecameric vanadium-dependent bromoperoxidase from the red algae Corallina officinalis. J Mol Biol 299:1035–1049. doi:10.1006/jmbi.2000.3806

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Makde RD, Mahajan SK, Kumar V (2007) Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details. BioChemistry 46:2079–2090. doi:10.1021/bi062180g

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Bernard R, Joseph P, Guiseppi A et al (2003) YtsCD and YwoA, two independent systems that confer bacitracin resistance to Bacillus subtilis. FEMS Microbiol Lett 228:93–97

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Barreteau H, Magnet S, Ghachi M El et al (2009) Quantitative high-performance liquid chromatography analysis of the pool levels of undecaprenyl phosphate and its derivatives in bacterial membranes. J Chromatogr B Anal Technol Biomed Life Sci 877:213–220. doi:10.1016/j.jchromb.2008.12.010

    CAS  Article  Google Scholar 

  31. 31.

    Rost B, Yachdav G, Liu J (2004) The PredictProtein server. Nucleic Acids Res 32:W321–W326. doi:10.1093/nar/gkh377

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Touzé T, Blanot D, Mengin-Lecreulx D (2008) Substrate specificity and membrane topology of Escherichia coli PgpB, an undecaprenyl pyrophosphate phosphatase. J Biol Chem 283:16573–16583. doi:10.1074/jbc.M800394200

    Article  PubMed  Google Scholar 

  33. 33.

    Tatar LD, Marolda CL, Polischuk AN et al (2007) An Escherichia coli undecaprenyl-pyrophosphate phosphatase implicated in undecaprenyl phosphate recycling. Microbiology 153:2518–2529. doi:10.1099/mic.0.2007/006312-0

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Pompeo F, Van Heijenoort J, Mengin-Lecreulx D (1998) Probing the role of cysteine residues in glucosamine-1-phosphate acetyltransferase activity of the bifunctional glmU protein from Escherichia coli: Site-directed mutagenesis and characterization of the mutant enzymes. J Bacteriol 180:4799–4803

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917. doi:10.1139/o59-099

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Mascher T, Margulis NG, Wang T et al (2003) Cell wall stress responses in Bacillus subtilis: the regulatory network of the bacitracin stimulon. Mol Microbiol 50:1591–1604. doi:10.1046/j.1365-2958.2003.03786.x

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645. doi:10.1073/pnas.120163297

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Hamilton CM, Aldea M, Washburn BK et al (1989) New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol 171:4617–4622

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Baba T, Ara T, Hasegawa M et al (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2(2006):0008. doi:10.1038/msb4100050

    PubMed  Google Scholar 

  40. 40.

    Caffrey M, Cherezov V (2009) Crystallizing membrane proteins using lipidic mesophases. Nat Protoc 4:706–731. doi:10.1038/nprot.2009.31

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Cheng A, Hummel B, Qiu H, Caffrey M (1998) A simple mechanical mixer for small viscous lipid-containing samples. Chem Phys Lipids 95:11–21. doi:10.1016/S0009-3084(98)00060-7

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Li D, Boland C, Walsh K, Caffrey M (2012) Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases. J Vis Exp e4000. doi:10.3791/4000

  43. 43.

    Li D, Boland C, Aragao D, et al (2012) Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography. J Vis Exp e4001. doi:10.3791/4001

  44. 44.

    Kabsch W (2010) XDS. Acta Crystallogr Sect D Biol Crystallogr 66:125–132. doi:10.1107/S0907444909047337

    CAS  Article  Google Scholar 

  45. 45.

    Terwilliger TC, Adams PD, Read RJ et al (2009) Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr Sect D Biol Crystallogr 65:582–601. doi:10.1107/S0907444909012098

    CAS  Article  Google Scholar 

  46. 46.

    Afonine P V., Grosse-Kunstleve RW, Echols N et al (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr Sect D Biol Crystallogr 68:352–367. doi:10.1107/S0907444912001308

    CAS  Article  Google Scholar 

  47. 47.

    Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr Sect D Biol Crystallogr 66:486–501. doi:10.1107/S0907444910007493

    CAS  Article  Google Scholar 

  48. 48.

    Afonine PV, Moriarty NW, Mustyakimov M, et al (2015) FEM: feature-enhanced map. Acta Crystallogr Sect D Biol Crystallogr 71:646–666. doi:10.1107/S1399004714028132

    CAS  Article  Google Scholar 

  49. 49.

    Krieger E, Darden T, Nabuurs SB et al (2004) Making optimal use of empirical energy functions: force-field parameterization in crystal space. Proteins Struct Funct Genet 57:678–683. doi:10.1002/prot.20251

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Best RB, Hummer G (2009) Optimized molecular dynamics force fields applied to the helix-coil transition of polypeptides. J Phys Chem B 113:9004–9015. doi:10.1021/jp901540t

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Essmann U, Perera L, Berkowitz ML et al (1995) A smooth particle mesh Ewald method. J Chem Phys 103:8577–8593

    CAS  Article  Google Scholar 

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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 or Martin Caffrey or Frédéric Kerff.

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The authors declare no competing financial interests.

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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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Keywords

  • Membrane protein structure
  • Bacterial lipids metabolism
  • Undecaprenyl phosphate
  • Peptidoglycan-related lipid