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New Insight into the Catalytic Mechanism of Bacterial MraY from Enzyme Kinetics and Docking Studies*

  • Author Footnotes
    1 Supported by ZonMW Grant 205.100.008 from the Netherlands Organization for Scientific Research (NWO).
    Yao Liu
    Footnotes
    1 Supported by ZonMW Grant 205.100.008 from the Netherlands Organization for Scientific Research (NWO).
    Affiliations
    Institute of Biomembranes, Department of Membrane Biochemistry and Biophysics, Utrecht University, 3584 CH, Utrecht, the Netherlands
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  • Author Footnotes
    1 Supported by ZonMW Grant 205.100.008 from the Netherlands Organization for Scientific Research (NWO).
    垚 刘
    Footnotes
    1 Supported by ZonMW Grant 205.100.008 from the Netherlands Organization for Scientific Research (NWO).
    Affiliations
    Institute of Biomembranes, Department of Membrane Biochemistry and Biophysics, Utrecht University, 3584 CH, Utrecht, the Netherlands
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  • João P.G.L.M. Rodrigues
    Affiliations
    Department of Computational Structural Biology, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH, Utrecht, the Netherlands
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  • Alexandre M.J.J. Bonvin
    Affiliations
    Department of Computational Structural Biology, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH, Utrecht, the Netherlands
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  • Esther A. Zaal
    Affiliations
    Departments of Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH, Utrecht, the Netherlands
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  • Author Footnotes
    2 Supported by a VENI grant (project 722.013.009) from the NWO.
    Celia R. Berkers
    Footnotes
    2 Supported by a VENI grant (project 722.013.009) from the NWO.
    Affiliations
    Departments of Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH, Utrecht, the Netherlands
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  • Michal Heger
    Affiliations
    Department of Experimental Surgery, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands
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  • Katarzyna Gawarecka
    Affiliations
    Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
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  • Ewa Swiezewska
    Affiliations
    Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
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  • Eefjan Breukink
    Correspondence
    To whom correspondence should be addressed: Membrane Biochemistry and Biophysics, Institute of Biomembranes, Utrecht University, 3584 CH, Utrecht, the Netherlands. Tel.:31-30-2533523;
    Affiliations
    Institute of Biomembranes, Department of Membrane Biochemistry and Biophysics, Utrecht University, 3584 CH, Utrecht, the Netherlands
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  • Maarten R. Egmond
    Affiliations
    Institute of Biomembranes, Department of Membrane Biochemistry and Biophysics, Utrecht University, 3584 CH, Utrecht, the Netherlands
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  • Author Footnotes
    * The authors declare that they have no conflicts of interest with the contents of this article.
    1 Supported by ZonMW Grant 205.100.008 from the Netherlands Organization for Scientific Research (NWO).
    2 Supported by a VENI grant (project 722.013.009) from the NWO.
Open AccessPublished:May 18, 2016DOI:https://doi.org/10.1074/jbc.M116.717884
      Phospho-MurNAc-pentapeptide translocase (MraY) catalyzes the synthesis of Lipid I, a bacterial peptidoglycan precursor. As such, MraY is essential for bacterial survival and therefore is an ideal target for developing novel antibiotics. However, the understanding of its catalytic mechanism, despite the recently determined crystal structure, remains limited. In the present study, the kinetic properties of Bacillus subtilis MraY (BsMraY) were investigated by fluorescence enhancement using dansylated UDP-MurNAc-pentapeptide and heptaprenyl phosphate (C35-P, short-chain homolog of undecaprenyl phosphate, the endogenous substrate of MraY) as second substrate. Varying the concentrations of both of these substrates and fitting the kinetics data to two-substrate models showed that the concomitant binding of both UDP-MurNAc-pentapeptide-DNS and C35-P to the enzyme is required before the release of the two products, Lipid I and UMP. We built a model of BsMraY and performed docking studies with the substrate C35-P to further deepen our understanding of how MraY accommodates this lipid substrate. Based on these modeling studies, a novel catalytic role was put forward for a fully conserved histidine residue in MraY (His-289 in BsMraY), which has been experimentally confirmed to be essential for MraY activity. Using the current model of BsMraY, we propose that a small conformational change is necessary to relocate the His-289 residue, such that the translocase reaction can proceed via a nucleophilic attack of the phosphate moiety of C35-P on bound UDP-MurNAc-pentapeptide.

      Introduction

      Among the enzymes involved in bacterial peptidoglycan synthesis, phospho-N-acetylmuramyl-pentapeptide translocase (MraY
      The abbreviations used are: MraY, phospho-MurNAc-pentapeptide translocase; BsMraY, Bacillus subtilis MraY; AaMraY, Aquifex aeolicus MraY; UDP-MurNAc-pentapeptide or UMpp, UDP-N-acetylmuramic acid-pentapeptide; C35-P, heptaprenyl phosphate; C55-P, undecaprenyl phosphate; DDM, N-dodecyl-β-d-maltopyranoside; [15N2]UMP, uridine-[15N2]5′-monophosphate; TM, transmembrane helix.
      ; EC 2.7.8.13) has been studied extensively (
      • Lloyd A.J.
      • Brandish P.E.
      • Gilbey A.M.
      • Bugg T.D.H.
      Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli: catalytic role of conserved aspartic acid residues.
      ,
      • Lovering A.L.
      • Safadi S.S.
      • Strynadka N.C.J.
      Structural perspective of peptidoglycan biosynthesis and assembly.
      ). This enzyme performs the initial membrane step in this process, forming undecaprenyl-N-acetylmuramyl-pentapeptide (Lipid I) from UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) and undecaprenyl phosphate, in both Gram-positive and Gram-negative bacteria. Given the role of MraY in bacterial cell wall synthesis (
      • Mihalyi A.
      • Jamshidi S.
      • Slikas J.
      • Bugg T.D.H.
      Identification of novel inhibitors of phospho-MurNAc-pentapeptide translocase MraY from library screening: isoquinoline alkaloid michellamine B and xanthene dye phloxine B.
      ,
      • Winn M.
      • Goss R.J.M.
      • Kimura K.-I.
      • Bugg T.D.H.
      Antimicrobial nucleoside antibiotics targeting cell wall assembly: recent advances in structure-function studies and nucleoside biosynthesis.
      ) and cell growth (
      • Boyle D.S.
      • Donachie W.D.
      mraY is an essential gene for cell growth in Escherichia coli.
      ), this enzyme is an interesting target for antibacterial drugs. Recently, the crystal structure of MraY from the Gram-negative species Aquifex aeolicus (Protein Data Bank entry 4J72) was determined (
      • Chung B.C.
      • Zhao J.
      • Gillespie R.A.
      • Kwon D.-Y.
      • Guan Z.
      • Hong J.
      • Zhou P.
      • Lee S.-Y.
      Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.
      ). The enzyme was extracted from its membrane environment with detergent and crystallized as a symmetrical homodimer. Each protomer consists of 10 transmembrane helices, with both the N and C termini locating on the periplasmic side (outside) of the cytoplasmic membrane (
      • Lloyd A.J.
      • Brandish P.E.
      • Gilbey A.M.
      • Bugg T.D.H.
      Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli: catalytic role of conserved aspartic acid residues.
      ). Before the publication of this high resolution (3.3 Å) structure, other studies attempted to unravel the catalytic mechanism of action of MraY by site-directed mutagenesis and kinetics studies, using either membrane-embedded MraY or detergent-extracted and purified preparations (
      • Lloyd A.J.
      • Brandish P.E.
      • Gilbey A.M.
      • Bugg T.D.H.
      Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli: catalytic role of conserved aspartic acid residues.
      ,
      • Bouhss A.
      • Crouvoisier M.
      • Blanot D.
      • Mengin-Lecreulx D.
      Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis.
      ,
      • Al-Dabbagh B.
      • Henry X.
      • El Ghachi M.
      • Auger G.
      • Blanot D.
      • Parquet C.
      • Mengin-Lecreulx D.
      • Bouhss A.
      Active site mapping of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, catalyzing the first membrane step of peptidoglycan biosynthesis.
      ). These studies proposed that catalysis proceeds most likely via a one-step process, although a two-step process has also been suggested (
      • Lloyd A.J.
      • Brandish P.E.
      • Gilbey A.M.
      • Bugg T.D.H.
      Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli: catalytic role of conserved aspartic acid residues.
      ). In the single-step process, a ternary complex of MraY, UDP-MurNAc-pentapeptide, and undecaprenyl phosphate (C55-P) yields Lipid I with concomitant release of UMP. In the two-step process, UMP is released, yielding a covalently bound phospho-MurNAc-pentapeptide intermediate that is subsequently attacked by C55-P to produce Lipid I. However, no direct experimental evidence was provided for either proposal. The kinetics values of MraY for its nucleotide and lipid substrates that have been reported in literature so far are not consistent with each other. Bouhss et al. (
      • Bouhss A.
      • Crouvoisier M.
      • Blanot D.
      • Mengin-Lecreulx D.
      Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis.
      ) obtained the Km value of MraY by varying the concentration of one substrate while keeping the other at a fixed value. The authors reported apparent Km values for UDP-MurNAc-pentapeptide and C55-P of 1.0 ± 0.3 and 0.16 ± 0.08 mm, respectively. This result was later challenged by another study (
      • Ma Y.
      • Münch D.
      • Schneider T.
      • Sahl H.-G.
      • Bouhss A.
      • Ghoshdastider U.
      • Wang J.
      • Dötsch V.
      • Wang X.
      • Bernhard F.
      Preparative scale cell-free production and quality optimization of MraY homologues in different expression modes.
      ) reporting an apparent Km value for UDP-MurNAc-pentapeptide at 36.2 ± 3.6 μm. The major difference between these two studies is the concentration of C55-P used in the reaction, namely 1.1 mm (
      • Bouhss A.
      • Crouvoisier M.
      • Blanot D.
      • Mengin-Lecreulx D.
      Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis.
      ) and 50 μm (
      • Ma Y.
      • Münch D.
      • Schneider T.
      • Sahl H.-G.
      • Bouhss A.
      • Ghoshdastider U.
      • Wang J.
      • Dötsch V.
      • Wang X.
      • Bernhard F.
      Preparative scale cell-free production and quality optimization of MraY homologues in different expression modes.
      ). This indicates that the concentration of both substrates should be varied to allow determination of the true Km value. In the present study, we performed more extensive kinetics studies on the detergent-solubilized MraY. Pure heptaprenyl phosphate (C35-P) was used as the preferred lipid substrate throughout our study. Other MraY studies (
      • Brandish P.E.
      • Burnham M.K.
      • Lonsdale J.T.
      • Southgate R.
      • Inukai M.
      • Bugg T.D.H.
      Slow binding inhibition of phospho-N-acetylmuramyl-pentapeptide-translocase (Escherichia coli) by mureidomycin A.
      ,
      • Rodolis M.T.
      • Mihalyi A.
      • O'Reilly A.
      • Slikas J.
      • Roper D.I.
      • Hancock R.E.W.
      • Bugg T.D.H.
      Identification of a novel inhibition site in translocase MraY based upon the site of interaction with lysis protein e from bacteriophage ?X174.
      ) have also reported the use of C35-P instead of the natural lipid substrate C55-P. Polyprenyl phosphates with shorter chain lengths are also accepted as substrates by MraY (
      • Huang L.Y.
      • Huang S.H.
      • Chang Y.C.
      • Cheng W.C.
      • Cheng T.J.
      • Wong C.H.
      Enzymatic synthesis of lipid II and analogues.
      ), but to the best of our knowledge, the exact effect of the prenyl chain length on the activity of MraY has not yet been studied in detail. Together with kinetic studies, we built a model for MraY from Bacillus subtilis, carried out docking experiments with C35-P, and analyzed the conserved residues in both Gram-positive and Gram-negative MraY species. The shortened chain length of C35-P compared with C55-P makes the docking results more reliable because of the reduced number of possible conformations of the prenyl chain. Many algorithms/programs are available for predicting protein-substrate binding. The majority of these approaches, however, focus on soluble proteins in an aqueous environment because more experimental data are available. For our docking studies, HADDOCK (high ambiguity driven protein-protein docking) (
      • Dominguez C.
      • Boelens R.
      • Bonvin A.M.J.J.
      HADDOCK: a protein-protein docking approach based on biochemical or biophysical information.
      ), an approach developed for protein complex docking based on biochemical and/or biophysical interaction data, was used. Unlike other docking methods, HADDOCK uses ambiguous interaction restraints to drive the docking. The docked structures are given a HADDOCK score, after calculations, according to their intermolecular energy, namely a weighted sum of desolvation, van der Waals, electrostatic, and ambiguous interaction restraint energy terms. In our case, the default desolvation energy term for aqueous protein docking was neglected, and a novel “z-restraint” was introduced to keep our model in the right orientation in the simulated membranes. Together, our findings provided a novel concept for the development of MraY inhibitors and imply that blocking the binding of the lipid substrate to the enzyme, by targeting His-298, may be a viable approach. This is of great interest, given that the inhibitor development for MraY has not been very successful so far.

      Author Contributions

      Y. L., E. B., and M. R. E. developed this study and designed the experiments; Y. L. carried out the production and activity study of MraY and its mutants; J. P. G. L. M. R. and A. M. J. J. B. carried out the modeling and docking studies; E. A. Z. and C. R. B. carried out mass spectrometry measurements and data analyses; M. H. carried out partial kinetics study and data analyses; and K. G. and E. S. synthesized C55-P and C35-P. All authors reviewed the results, revised the manuscript, and approved the final version of the manuscript.

      Acknowledgments

      We thank Dr. Tamimount Mohammadi for critically reading and commenting on the manuscript and Elisabete Moura for assistance during some experimental work. We also thank Prof. Ry Young for providing the ΔslyD BL21 strain.

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