Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis

Ghachi, Meriem El; Howe, Nicole; Huang, Chia‐Ying; Oliéric, V.; Warshamanage, Rangana; Touzé, Thierry; Weichert, Dietmar; Stansfeld, Phillip J.; Wang, Meitian; Kerff, Frédéric; Caffrey, Martin

doi:10.1038/s41467-018-03477-5

Cited by 47 publications

(64 citation statements)

References 60 publications

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“…BacA represents the other type of C 55 -PP phosphatases, whose structure strongly suggested that the protein might display a C 55 -P flippase activity 7,10 . Such an activity is required to relocalize the C 55 -P product back to the inner side of the membrane to end recycling.…”

Section: Discussionmentioning

confidence: 99%

Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli

Tian

Auger

Manat

et al. 2020

Sci Rep

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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. Undecaprenyl phosphate (C 55-P) plays an essential role in the biogenesis of bacterial envelope polysaccharides such as the peptidoglycan 1. It is used as a lipid carrier allowing the translocation of polymer subunits across the plasma membrane to the outer site, where the polymers are assembled 2 (Supplementary Fig. S1). The synthesis of C 55-P proceeds via the hydrolysis of its precursor, the undecaprenyl pyrophosphate (C 55-PP), itself being de novo synthesized at the cytosolic side of the plasma membrane or released during subunits polymerization at the outer side (Supplementary Fig. S1) 1. Four integral membrane enzymes catalyzing C 55-PP hydrolysis have been identified in Escherichia coli: BacA, PgpB, YbjG and LpxT (formerly YeiU), which belong to two unrelated protein families: BacA and Phosphatidic Acid Phosphatases of type 2 (PAP2) 3-5. None of these enzymes is essential for growth, but the simultaneous knockout of bacA, pgpB and ybjG genes elicits a lethal phenotype due to a default of C 55-P supply 4. These enzymes have their active sites oriented towards the periplasm, suggesting they may rather be involved in C 55-PP recycling 5-10. The mechanism of translocation of C 55-P back to the inner side of the membrane is yet unknown. The structure of BacA, which is reminiscent to that of transporters, raised the hypothesis that it may also catalyze the flip of C 55-P 7,10. The lack of a known cytoplasm-oriented C 55-PP phosphatase raised also the question as to whether BacA and PAP2s are required for the dephosphorylation of de novo synthesized C 55-PP (Supplementary Fig. S1). PgpB is involved in another essential metabolic pathway, i.e. the synthesis of phosphatidylglyc...

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Section: Discussionmentioning

confidence: 99%

Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli

Tian

Auger

Manat

et al. 2020

Sci Rep

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“…The successful application of the IMISX method both at room and cryo‐temperatures was demonstrated with seven MPs including two transporters, PepT St and AlgE, four enzymes, DgkA, LspA, PgpB , BacA , and a GPCR, ß 2 AR . For lysozyme, exceptional data quality was obtained from IMISX by sulfur‐SAD phasing using 992 crystals .…”

Section: In Meso In Situ Serial X‐ray Crystallography (Imisx)mentioning

confidence: 99%

In situ crystallography as an emerging method for structure solution of membrane proteins: the case of CCR2A

Cheng¹,

Huang

Hennig³

et al. 2019

The FEBS Journal

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The in meso in situ serial X‐ray crystallization method (Huang et al., (2015) Acta Crystallogr D Biol Crystallogr 71, 1238) combines lipid cubic phase crystallization, direct freezing of the crystallization droplet without handling of the crystals, and data collection in situ. Recently, this method was used to overcome the mechanical fragility of crystals which enabled the X‐ray structure determination of chemokine receptor 2A (Apel et al., (2019) Structure 27, 427) at 2.7 Å resolution. The CCR2 structure provides the structural basis for ligand selectivity of CCR2 against chemokine receptor 5 and provides insights into the residence time of MK‐0812 analogs based on molecular dynamics simulations. These findings offer new opportunities for drug discovery targeting chemokine receptors.

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“…The cubic phase has also found application as a native-like medium in which to port crystals across beams for time-resolved serial crystallography at synchrotron (Nogly et al, 2015;Weinert et al, 2017) and free-electron laser X-ray sources (Weierstall et al, 2014;White et al, 2016;Nogly et al, 2018;Cheng, 2020). The recently developed in situ in meso serial X-ray crystallography (IMISX) method uses perforated double-stick tape to create wells for crystallization and supporting sachets (IMISX wells) in which to deliver the crystal-laden mesophase to the X-ray beam without the need for direct crystal harvesting (Huang et al, 2015(Huang et al, , 2016(Huang et al, , 2018El Ghachi et al, 2018;Apel et al, 2019;Cheng et al, 2019). The original IMISX method involved securing a slightly curved thin-film sachet to a pin on a goniometer (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…They provide for a wide scanning area and are suitable for post-crystallization treatments such as ligand and heavy-atom soaking (Li et al, 2015;Rucktooa et al, 2018;Huang et al, 2018). The holders can be used with a host of in situ window materials such as cyclic olefin polymer (COP) (Axford et al, 2016;Apel et al, 2019), cyclic olefin copolymer (COC) (Huang et al, 2015(Huang et al, , 2016El Ghachi et al, 2018), biaxially oriented polyethylene terephthalate (Mylar) (Broecker et al, 2016) and silicon nitride (Cherezov & Caffrey, 2007;Murray et al, 2015;Roedig et al, 2015;Owen et al, 2017). Furthermore, the holders are 3D printed and reusable.…”

Section: Introductionmentioning

confidence: 99%

3D-printed holders forin meso in situfixed-target serial X-ray crystallography

et al. 2020

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The in meso in situ serial X-ray crystallography method was developed to ease the handling of small fragile crystals of membrane proteins and for rapid data collection on hundreds of microcrystals directly in the growth medium without the need for crystal harvesting. To facilitate mounting of these in situ samples on a goniometer at cryogenic or at room temperatures, two new 3D-printed holders have been developed. They provide for cubic and sponge phase sample stability in the X-ray beam and are compatible with sample-changing robots. The holders can accommodate a variety of window material types, as well as bespoke samples for diffraction screening and data collection at conventional macromolecular crystallography beamlines. They can be used for convenient postcrystallization treatments such as ligand and heavy-atom soaking. The design, assembly and application of the holders for in situ serial crystallography are described. Files for making the holders using a 3D printer are included as supporting information.

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Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis

Cited by 47 publications

References 60 publications

Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli

Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli

In situ crystallography as an emerging method for structure solution of membrane proteins: the case of CCR2A

3D-printed holders forin meso in situfixed-target serial X-ray crystallography

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