Structural and Chemical Aspects of Resistance to the Antibiotic Fosfomycin Conferred by FosB from Bacillus cereus

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• 作者：Matthew K. Thompson ; Mary E. Keithly ; Joel Harp ; Paul D. Cook ; Kevin L. Jagessar ; Gary A. Sulikowski ; Richard N. Armstrong
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：October 15, 2013
• 年：2013
• 卷：52
• 期：41
• 页码：7350-7362
• 全文大小：646K
• 年卷期：v.52,no.41(October 15, 2013)
• ISSN：1520-4995

文摘

The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M²⁺-dependent thiol tranferases that catalyze nucleophilic addition of either lass="smallcaps">l-cysteine (lass="smallcaps">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 (FosB^Bc). The overall structure of FosB^Bc, at 1.27 脜 resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosB^Bc 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 FosB^Bc 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 lass="smallcaps">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 FosB^Bc in complex with the lass="smallcaps">l-Cys-fosfomycin product (1.55 脜 resolution) and in complex with the bacillithiol-fosfomycin product (1.77 脜 resolution) coordinated to a Mn²⁺ metal in the active site have been determined. The lass="smallcaps">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 FosB^Bc indicated that the enzyme has a preference for BSH over lass="smallcaps">l-Cys when activated by Mn²⁺ and is inhibited by Zn²⁺. The fact that Zn²⁺ is an inhibitor of FosB^Bc was used to obtain a ternary complex structure of the enzyme with both fosfomycin and lass="smallcaps">l-Cys bound.