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)

Stoyan K. Smoukov, Joshua Telser, Bryan A. Bernat, Chris L. Rife, Richard N. Armstrong*, Brian M. Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

49 Scopus citations

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-Mn2+ 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.

Original languageEnglish (US)
Pages (from-to)2318-2326
Number of pages9
JournalJournal of the American Chemical Society
Volume124
Issue number10
DOIs
StatePublished - Mar 13 2002

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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