| Abstract
| - FosA is a manganese metalloglutathione transferase that confers resistance to the broad-spectrumantibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid. The reaction catalyzed by FosA involves theattack 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. Thecoordination environment of the Mn(II) in the FosA·Mn2+ complex is composed of a glutamate and twohistidine ligands and three water molecules. Here we report EPR spectroscopic studies on FosA, in whichEPR spectra were obtained at 35 GHz and 2 K using dispersion-detection rapid-passage techniques. Thisapproach 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-fieldsplitting. 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 ofthe EPR spectra shows that binding of substrate or analogue causes a profound change in the electronicparameters of the Mn(II) ion. The axial zero-field splitting changes from |D| = 0.06 cm-1 for substrate-freeenzyme 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 analysisof this change indicates that binding of the phosphonate/phosphate group of substrate or analogue changesthe electronic energy levels of the Mn(II) 3d orbitals by several thousand cm-1, indicative of a significantchange 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 ofthe glutamate ligand.
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