title: Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway.
abstract: Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.
authors.affiliation: From the Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J. W. Goethe-University, 60438 Frankfurt-am-Main, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Henrich
authors.affiliation: From the Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J. W. Goethe-University, 60438 Frankfurt-am-Main, Germany, the School of Bioscience and Bioengineering, South China University of Technology, 510006 Guangzhou, China, bimayikobe@scut.edu.cn.
authors.forename: Yi
authors.initials: Y
authors.lastname: Ma
authors.affiliation: the Institute for Pharmaceutical Microbiology, University of Bonn, Germany, and the German Centre for Infection Research (DZIF), partner site Cologne-Bonn, 53115 Bonn, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Engels
authors.affiliation: the Institute for Pharmaceutical Microbiology, University of Bonn, Germany, and.
authors.forename: Daniela
authors.initials: D
authors.lastname: Münch
authors.affiliation: the Institute for Pharmaceutical Microbiology, University of Bonn, Germany, and.
authors.forename: Christian
authors.initials: C
authors.lastname: Otten
authors.affiliation: the Institute for Pharmaceutical Microbiology, University of Bonn, Germany, and the German Centre for Infection Research (DZIF), partner site Cologne-Bonn, 53115 Bonn, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Schneider
authors.affiliation: the Institute for Pharmaceutical Microbiology, University of Bonn, Germany, and.
authors.forename: Beate
authors.initials: B
authors.lastname: Henrichfreise
authors.affiliation: the Institute for Pharmaceutical Microbiology, University of Bonn, Germany, and the German Centre for Infection Research (DZIF), partner site Cologne-Bonn, 53115 Bonn, Germany.
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Sahl
authors.affiliation: From the Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J. W. Goethe-University, 60438 Frankfurt-am-Main, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.affiliation: From the Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J. W. Goethe-University, 60438 Frankfurt-am-Main, Germany, fbern@bpc.uni-frankfurt.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry*; D053898:Biosynthetic Pathways; D001886:Bordetella pertussis / Q000201:enzymology; D025065:Borrelia burgdorferi / Q000201:enzymology; D002473:Cell Wall / Q000737:chemistry; D002474:Cell-Free System; D016993:Chlamydophila pneumoniae / Q000201:enzymology; D003593:Cytoplasm / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005578:Fosfomycin / Q000737:chemistry; D016480:Helicobacter pylori / Q000201:enzymology; D008823:Micelles; D009005:Monosaccharides / Q000096:biosynthesis*; D009842:Oligopeptides / Q000096:biosynthesis*; D010455:Peptides / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D014166:Transferases / Q000737:chemistry*; D017855:Transferases (Other Substituted Phosphate Groups); D014415:Tunicamycin / Q000737:chemistry; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D008823:Micelles; D009005:Monosaccharides; D009842:Oligopeptides; D010455:Peptides; D010457:Peptidoglycan; D011506:Proteins; D011994:Recombinant Proteins; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C120179:feglymycin; C438940:lipid I; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; D014415:Tunicamycin; D005578:Fosfomycin; D004247:DNA; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria
keywords: cell-free expression; lipid II; lipid-protein interaction; membrane enzyme; nanodisc; peptidoglycan; protein folding; recombinant protein expression; synthetic biology