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Conformational Flexibility of Fosfomycin Resistance Protein Fosx from Listeria Monocytogenes. Svetlana Pakhomova^a, Kerry Fillgrove^b, Richard N. Armstrong^b and Marcia E. Newcomer^a, ^aDept. of Biological Sciences, Louisiana State Univ., Baton Rouge, LA 70803; ^bDept. of Biochemistry, Vanderbilt Univ. School of Medicine, Nashville, TN 37232-0146.

Fosfomycin resistance proteins (FosA, FosB and FosX) are both plasmid and genomically encoded enzymes that belong to the vicinal oxygen chelate superfamily of metalloenzymes. These enzymes confer resistance to the broad spectrum antibiotic fosfomycin [(1R,2S)-epoxypropylphosphonic acid]. They are functional as homodimers, require divalent cations for activation, share modest (23-40%) sequence identity between subclasses, but use different mechanism to inactivate fosfomycin. The structure of FosA has recently been solved¹. Listeria monocytogenes gene lmo1702 (FosX) was cloned, overexpressed in E. coli and subsequently purified using ion exchange and size exclusion chromatography. This novel enzyme catalyzes the addition of water to the epoxide ring of fosfomycin forming the diol 1,2-dihydroxypropyl-phosphonic acid. FosX requires a divalent cation for activation, with Mn²⁺ being the preferred metal.

Three crystal forms have been obtained for FosX and the structures have been solved by molecular replacement using FosA as a model (28% sequence identity). Significant conformational differences of the protein molecule are observed between crystal forms, depending on the pH of the crystallization buffer. The low pH structures show flexible character of several loops in the vicinity of the active site. In addition to significant movements of the loops, there is a dramatic change in the C-terminus in the high pH structure. The terminal helix (previously on the surface of the protein) is disrupted and fills the active site. In addition, E126 is coordinated to Mn²⁺. The significance and functional implication of these structural findings are discussed.