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Publication Abstract from PubMed

The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M2+-dependent thiol tranferases that catalyze nucleophilic addition of either l-cysteine (l-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bacteriacidal properties. Here we report the structural and functional characterization of FosB from Bacillus cereus (FosBBc). The overall structure of FosBBc, at 1.27 A resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosBBc cocrystallized with fosfomycin and a variety of divalent metals, including Ni2+, Mn2+, Co2+, and Zn2+, indicate that the antibiotic coordinates to the active site metal center in an orientation similar to that found in the structurally homologous manganese-dependent fosfomycin resistance enzyme, FosA. Surface analysis of the FosBBc structures show a well-defined binding pocket and an access channel to C1 of fosfomycin, the carbon to which nucleophilic addition of the thiol occurs. The pocket and access channel are appropriate in size and shape to accommodate l-Cys or BSH. Further investigation of the structures revealed that the fosfomycin molecule, anchored by the metal, is surrounded by a cage of amino acids that hold the antibiotic in an orientation such that C1 is centered at the end of the solvent channel, positioning the compound for direct nucleophilic attack by the thiol substrate. In addition, the structures of FosBBc in complex with the l-Cys-fosfomycin product (1.55 A resolution) and in complex with the bacillithiol-fosfomycin product (1.77 A resolution) coordinated to a Mn2+ metal in the active site have been determined. The l-Cys moiety of either product is located in the solvent channel, where the thiol has added to the backside of fosfomycin C1 located at the end of the channel. Concomitant kinetic analyses of FosBBc indicated that the enzyme has a preference for BSH over l-Cys when activated by Mn2+ and is inhibited by Zn2+. The fact that Zn2+ is an inhibitor of FosBBc was used to obtain a ternary complex structure of the enzyme with both fosfomycin and l-Cys bound.

Structural and Chemical Aspects of Resistance to the Antibiotic Fosfomycin Conferred by FosB from Bacillus cereus.,Thompson MK, Keithly ME, Harp J, Cook PD, Jagessar KL, Sulikowski GA, Armstrong RN Biochemistry. 2013 Sep 30. PMID:24004181^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.