title: EPR study of substrate binding to the Mn(II) active site of the bacterial antibiotic resistance enzyme FosA: a better way to examine Mn(II).
abstract: FosA is a manganese metalloglutathione transferase that confers resistance to the broad-spectrum antibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid. The reaction catalyzed by FosA involves the attack by glutathione on fosfomycin to yield the product 1-(S-glutathionyl)-2-hydroxypropylphosphonic acid. The enzyme is a dimer of 16 kDa subunits, each of which harbors one mononuclear Mn(II) site. The coordination environment of the Mn(II) in the FosA x Mn(2+) complex is composed of a glutamate and two histidine ligands and three water molecules. Here we report EPR spectroscopic studies on FosA, in which EPR spectra were obtained at 35 GHz and 2 K using dispersion-detection rapid-passage techniques. This approach provides an absorption envelope line shape, in contrast to the conventional (slow-passage) derivative line shape, and is a more reliable way to collect spectra from Mn(II) centers with large zero-field splitting. We obtain excellent spectra of FosA bound with substrate, substrate analogue phosphate ion, and product, whereas these states cannot be studied by X-band, slow-passage methods. Simulation of the EPR spectra shows that binding of substrate or analogue causes a profound change in the electronic parameters of the Mn(II) ion. The axial zero-field splitting changes from [D] = 0.06 cm(-1) for substrate-free enzyme to 0.23 cm(-1) for fosfomycin-bound enzyme, 0.28 (1) cm(-1) for FosA with phosphate, and 0.27 (1) cm(-1) with product. Such a large zero-field splitting is uncommon for Mn(II). A simple ligand field analysis of this change indicates that binding of the phosphonate/phosphate group of substrate or analogue changes the electronic energy levels of the Mn(II) 3d orbitals by several thousand cm(-1), indicative of a significant change in the Mn(II) coordination sphere. Comparison with related enzymes (glyoxalase I and MnSOD) suggests that the change in the coordination environment on substrate binding may correspond to loss of the glutamate ligand.
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, USA.
authors.forename: Stoyan K
authors.initials: SK
authors.lastname: Smoukov
authors.forename: Joshua
authors.initials: J
authors.lastname: Telser
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Bernat
authors.forename: Chris L
authors.initials: CL
authors.lastname: Rife
authors.forename: Richard N
authors.initials: RN
authors.lastname: Armstrong
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins; D001665:Binding Sites; D024881:Drug Resistance, Bacterial; D004578:Electron Spin Resonance Spectroscopy; D005578:Fosfomycin / Q000737:chemistry* / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005578:Fosfomycin; D008345:Manganese; D005982:Glutathione Transferase