title: REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant.
abstract: The peptidoglycan of Gram-positive bacteria consists of glycan chains with attached short peptide stems cross-linked to one another by glycyl bridges. The bridge of Staphylococcus aureus has five glycyl units and that of its FemA mutant has one. These long- and short-bridge cross-links create totally different cell-wall architectures. S. aureus and its FemA mutant grown in the presence of an alanine-racemase inhibitor were labeled with d-[1-¹³C]alanine, l-[3-¹³C]alanine, [2-¹³C]glycine, and l-[5-¹⁹F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between ¹³C-labeled alanines and ¹⁹F-labeled lysine incorporated in the peptidoglycan. The alanyl ¹³C labels in the parent strain were preselected for C{F} and C{P} REDOR measurement by their proximity to the glycine label using ¹³C¹³C spin diffusion. The observed ¹³C¹³C and ¹³C³¹P distances are consistent with a tightly packed architecture containing only parallel stems in a repeating structural motif within the peptidoglycan. Dante selection of d-alanine and l-alanine frequencies followed by ¹³C¹³C spin diffusion rules out scrambling of carbon labels. Cell walls of FemA were also labeled by a combination of d-[1-¹³C]alanine and l-[¹⁵N]alanine. Proximity of chains was measured by C{N} and N{C} REDOR distances and asymptotic plateaus, and both were consistent with a mixed-geometry model. Binding of an ¹⁹F-labeled eremomycin analog in the FemA cell wall matches that of binding to the parent-strain cell wall and reveals the proximity of parallel stems in the alternating parallel-perpendicular mixed-geometry model for the FemA peptidoglycan lattice.
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO 63130, USA.
authors.forename: Manmilan
authors.initials: M
authors.lastname: Singh
authors.affiliation: Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76706, USA.
authors.forename: Sung Joon
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO 63130, USA.
authors.forename: Shasad
authors.initials: S
authors.lastname: Sharif
authors.affiliation: Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, Moscow 119021, Russia.
authors.forename: Maria
authors.initials: M
authors.lastname: Preobrazhenskaya
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO 63130, USA. Electronic address: jschaefer@wustl.edu.
authors.forename: Jacob
authors.initials: J
authors.lastname: Schaefer
mesh_terms: D000411:Alanine Racemase / Q000037:antagonists & inhibitors; D001426:Bacterial Proteins / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009154:Mutation; D010457:Peptidoglycan / Q000096:biosynthesis / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; C096686:FemA protein, Bacteria; D010457:Peptidoglycan; D000411:Alanine Racemase
keywords: Alanine racemase; Bacterial cell-walls; Solid-state NMR