[en] Ubiquitous PAP2 lipid phosphatases are involved in a wide array of central physiological functions. PgpB from Escherichia coli constitutes the archetype of this subfamily of membrane proteins. It displays a dual function by catalyzing the biosynthesis of two essential lipids, the phosphatidylglycerol (PG) and the undecaprenyl phosphate (C(55)-P). C(55)-P constitutes a lipid carrier allowing the translocation of peptidoglycan subunits across the plasma membrane. PG and C(55)-P are synthesized in a redundant manner by PgpB and other PAP2 and/or unrelated membrane phosphatases. Here, we show that PgpB is the sole, among these multiple phosphatases, displaying this dual activity. The inactivation of PgpB does not confer any apparent growth defect, but its inactivation together with another PAP2 alters the cell envelope integrity increasing the susceptibility to small hydrophobic compounds. Evidence is also provided of an interplay between PAP2s and the peptidoglycan polymerase PBP1A. In contrast to PGP hydrolysis, which relies on a His/Asp/His catalytic triad of PgpB, the mechanism of C(55)-PP hydrolysis appeared as only requiring the His/Asp diad, which led us to hypothesize distinct processes. Moreover, thermal stability analyses highlighted a substantial structural change upon phosphate binding by PgpB, supporting an induced-fit model of action.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Tian, Xudong
Auger, Rodolphe
Manat, Guillaume
Kerff, Frédéric ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'ingénierie des protéines
Mengin-Lecreulx, Dominique
Touzé, Thierry
Language :
English
Title :
Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli.
Manat, G. et al. Deciphering the metabolism of undecaprenyl-phosphate: The bacterial cell-wall unit carrier at the membrane frontier. Microb. Drug Resist. Larchmt. N 20, 199–214 (2014). DOI: 10.1089/mdr.2014.0035
Bouhss, A., Trunkfield, A. E., Bugg, T. D. H. & Mengin-Lecreulx, D. The biosynthesis of peptidoglycan lipid-linked intermediates. FEMS Microbiol. Rev. 32, 208–233 (2008). DOI: 10.1111/j.1574-6976.2007.00089.x
El Ghachi, M., Bouhss, A., Blanot, D. & Mengin-Lecreulx, D. The bacA gene of Escherichia coli encodes an undecaprenyl pyrophosphate phosphatase activity. J. Biol. Chem. 279, 30106–30113 (2004). DOI: 10.1074/jbc.M401701200
El Ghachi, M., Derbise, A., Bouhss, A. & Mengin-Lecreulx, D. Identification of multiple genes encoding membrane proteins with undecaprenyl pyrophosphate phosphatase (UppP) activity in Escherichia coli. J. Biol. Chem. 280, 18689–18695 (2005). DOI: 10.1074/jbc.M412277200
Touzé, T., Tran, A. X., Hankins, J. V., Mengin-Lecreulx, D. & Trent, M. S. Periplasmic phosphorylation of lipid A is linked to the synthesis of undecaprenyl phosphate. Mol. Microbiol. 67, 264–277 (2008). DOI: 10.1111/j.1365-2958.2007.06044.x
Touzé, T., Blanot, D. & Mengin-Lecreulx, D. Substrate specificity and membrane topology of Escherichia coli PgpB, an undecaprenyl pyrophosphate phosphatase. J. Biol. Chem. 283, 16573–16583 (2008). DOI: 10.1074/jbc.M800394200
El Ghachi, M. et al. Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis. Nat. Commun. 9, 1078 (2018). DOI: 10.1038/s41467-018-03477-5
Tatar, L. D., Marolda, C. L., Polischuk, A. N., van Leeuwen, D. & Valvano, M. A. An Escherichia coli undecaprenyl-pyrophosphate phosphatase implicated in undecaprenyl phosphate recycling. Microbiol. Read. Engl. 153, 2518–2529 (2007). DOI: 10.1099/mic.0.2007/006312-0
Manat, G. et al. Membrane topology and biochemical characterization of the Escherichia coli BacA undecaprenyl-pyrophosphate phosphatase. PLoS ONE 10, e0142870 (2015). DOI: 10.1371/journal.pone.0142870
Workman, S. D., Worrall, L. J. & Strynadka, N. C. J. Crystal structure of an intramembranal phosphatase central to bacterial cell-wall peptidoglycan biosynthesis and lipid recycling. Nat. Commun. 9, 1159 (2018). DOI: 10.1038/s41467-018-03547-8
Dillon, D. A. et al. The Escherichia coli pgpB gene encodes for a diacylglycerol pyrophosphate phosphatase activity. J. Biol. Chem. 271, 30548–30553 (1996). DOI: 10.1074/jbc.271.48.30548
Lu, Y.-H., Guan, Z., Zhao, J. & Raetz, C. R. H. Three phosphatidylglycerol-phosphate phosphatases in the inner membrane of Escherichia coli. J. Biol. Chem. 286, 5506–5518 (2011). DOI: 10.1074/jbc.M110.199265
Fan, J., Jiang, D., Zhao, Y., Liu, J. & Zhang, X. C. Crystal structure of lipid phosphatase Escherichia coli phosphatidylglycerophosphate phosphatase B. Proc. Natl. Acad. Sci. 111, 7636–7640 (2014). DOI: 10.1073/pnas.1403097111
Tong, S. et al. Structural insight into substrate selection and catalysis of lipid phosphate phosphatase PgpB in the cell membrane. J. Biol. Chem. 291, 18342–18352 (2016). DOI: 10.1074/jbc.M116.737874
Stukey, J. & Carman, G. M. Identification of a novel phosphatase sequence motif. Protein Sci. Publ. Protein Soc. 6, 469–472 (1997). DOI: 10.1002/pro.5560060226
Neuwald, A. F. An unexpected structural relationship between integral membrane phosphatases and soluble haloperoxidases. Protein Sci. Publ. Protein Soc. 6, 1764–1767 (1997). DOI: 10.1002/pro.5560060817
Ishikawa, K., Mihara, Y., Gondoh, K., Suzuki, E. & Asano, Y. X-ray structures of a novel acid phosphatase from Escherichia blattae and its complex with the transition-state analog molybdate. EMBO J. 19, 2412–2423 (2000). DOI: 10.1093/emboj/19.11.2412
Makde, R. D., Mahajan, S. K. & Kumar, V. Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details. Biochemistry 46, 2079–2090 (2007). DOI: 10.1021/bi062180g
Ghosh, A., Shieh, J.-J., Pan, C.-J., Sun, M.-S. & Chou, J. Y. The catalytic center of glucose-6-phosphatase. HIS176 is the nucleophile forming the phosphohistidine-enzyme intermediate during catalysis. J. Biol. Chem. 277, 32837–32842 (2002). DOI: 10.1074/jbc.M201853200
Ghosh, A., Shieh, J.-J., Pan, C.-J. & Chou, J. Y. Histidine 167 is the phosphate acceptor in glucose-6-phosphatase-beta forming a phosphohistidine enzyme intermediate during catalysis. J. Biol. Chem. 279, 12479–12483 (2004). DOI: 10.1074/jbc.M313271200
Ghachi, M. E. et al. Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis. Cell. Mol. Life Sci. CMLS 74, 2319–2332 (2017). DOI: 10.1007/s00018-017-2464-6
Zapun, A., Contreras-Martel, C. & Vernet, T. Penicillin-binding proteins and beta-lactam resistance. FEMS Microbiol. Rev. 32, 361–385 (2008). DOI: 10.1111/j.1574-6976.2007.00095.x
Meeske, A. J. et al. SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature 537, 634–638 (2016). DOI: 10.1038/nature19331
Yousif, S. Y., Broome-Smith, J. K. & Spratt, B. G. Lysis of Escherichia coli by beta-lactam antibiotics: deletion analysis of the role of penicillin-binding proteins 1A and 1B. J. Gen. Microbiol. 131, 2839–2845 (1985).
Hernández-Rocamora, V. M. et al. Coupling of polymerase and carrier lipid phosphatase prevents product inhibition in peptidoglycan synthesis. Cell Surf. 2, 1–13 (2018). DOI: 10.1016/j.tcsw.2018.04.002
Ariga, K. et al. Monolayer studies of single-chain polyprenyl phosphates. Langmuir ACS J. Surf. Colloids 21, 4578–4583 (2005). DOI: 10.1021/la0467887
Nicolaes, V. et al. Insights into the function of YciM, a heat shock membrane protein required to maintain envelope integrity in Escherichia coli. J. Bacteriol. 196, 300–309 (2014). DOI: 10.1128/JB.00921-13
Mahalakshmi, S., Sunayana, M. R., SaiSree, L. & Reddy, M. yciM is an essential gene required for regulation of lipopolysaccharide synthesis in Escherichia coli. Mol. Microbiol. 91, 145–157 (2014). DOI: 10.1111/mmi.12452
Klein, G., Kobylak, N., Lindner, B., Stupak, A. & Raina, S. Assembly of lipopolysaccharide in Escherichia coli requires the essential LapB heat shock protein. J. Biol. Chem. 289, 14829–14853 (2014). DOI: 10.1074/jbc.M113.539494
Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006). DOI: 10.1038/msb4100050
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. 97, 6640–6645 (2000). DOI: 10.1073/pnas.120163297
Hugonnet, J.-E. et al. Factors essential for l,d-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli. eLife 5, e19469 (2016). DOI: 10.7554/eLife.19469
Pompeo, F., van Heijenoort, J. & Mengin-Lecreulx, D. 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 (1998).
Crawford, R. W. et al. Very long O-antigen chains enhance fitness during Salmonella-induced colitis by increasing bile resistance. PLoS Pathog. 8, e1002918 (2012).
Tsai, C. M. & Frasch, C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119, 115–119 (1982).
Gasiorowski, E. et al. HupA, the main undecaprenyl pyrophosphate and phosphatidylglycerol phosphate phosphatase in Helicobacter pylori is essential for colonization of the stomach. PLoS Pathog. 15, e1007972 (2019).
Pagano, B. et al. Differential scanning calorimetry to investigate G-quadruplexes structural stability. Methods San Diego Calif. 64, 43–51 (2013).
