title: New Insight into the Catalytic Mechanism of Bacterial MraY from Enzyme Kinetics and Docking Studies.
abstract: 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.
authors.affiliation: From Institute of Biomembranes, Department of Membrane Biochemistry and Biophysics, Utrecht University, 3584 CH, Utrecht, the Netherlands.
authors.forename: Yao
authors.initials: Y
authors.lastname: Liu
authors.affiliation: the Departments of Computational Structural Biology and.
authors.forename: João P G L M
authors.initials: JP
authors.lastname: Rodrigues
authors.affiliation: the Departments of Computational Structural Biology and.
authors.forename: Alexandre M J J
authors.initials: AM
authors.lastname: Bonvin
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH, Utrecht, the Netherlands.
authors.forename: Esther A
authors.initials: EA
authors.lastname: Zaal
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH, Utrecht, the Netherlands.
authors.forename: Celia R
authors.initials: CR
authors.lastname: Berkers
authors.affiliation: the Department of Experimental Surgery, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands, and.
authors.forename: Michal
authors.initials: M
authors.lastname: Heger
authors.affiliation: the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Gawarecka
authors.affiliation: the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Swiezewska
authors.affiliation: From Institute of Biomembranes, Department of Membrane Biochemistry and Biophysics, Utrecht University, 3584 CH, Utrecht, the Netherlands, E.J.Breukink@uu.nl.
authors.forename: Eefjan
authors.initials: E
authors.lastname: Breukink
authors.affiliation: From Institute of Biomembranes, Department of Membrane Biochemistry and Biophysics, Utrecht University, 3584 CH, Utrecht, the Netherlands.
authors.forename: Maarten R
authors.initials: MR
authors.lastname: Egmond
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D007700:Kinetics; D008958:Models, Molecular; D009005:Monosaccharides / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups); D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D014542:Uridine Monophosphate / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009005:Monosaccharides; D009842:Oligopeptides; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C438940:lipid I; C002225:UDP-N-acetylmuramic acid pentapeptide; D014542:Uridine Monophosphate; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria
keywords: HADDOCK; MraY; docking; enzyme; helices; kinetics; mechanism; phosphate; structural model; structure-function