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

Abstract

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 Å resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosBBc co-crystallized 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-cysteine-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn2+ metal in the active site have been determined. The L-cysteine 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 bacillithiol over L-cysteine 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-cysteine bound.

Bacillithiol: a key protective thiol inStaphylococcusaureus

“…FosB is a fosfomycin resistance protein found in Firmicutes that was initially thought to catalyze the reaction of cysteine with the epoxide moiety of fosfomycin, a cell wall inhibitor [45]. However, after the discovery of bacillithiol, several biochemical studies found that bacillithiol is the preferred FosB thiol cosubstrate for fosfomycin detoxification, making FosB the first bacillithiol transferase to be described (see “Bacillithiol Transferases” section below) [26,46,47]. The importance of bacillithiol in fosfomycin detoxification was confirmed in B. subtilis [22,27,44] and B. anthracis Sterne [30] as mutants lacking bacillithiol were as sensitive to fosfomycin as the fosB mutant.…”

A Structural, Functional, and Computational Analysis of BshA, the First Enzyme in the Bacillithiol Biosynthesis Pathway

“…3–8 BSH is the preferred cosubstrate for FosB, an enzyme produced by certain species of Gram-positive bacteria that enables resistance to the FDA-approved antibiotic fosfomycin. 9–11 Sold under the trade name Monurol, fosfomycin is the only clinically-used drug that inhibits MurA, the enzyme that catalyzes the committed step in bacterial cell wall biogenesis. 12 Fosfomycin-resistant organisms in which the BSH biosynthesis genes have been disrupted demonstrate increased sensitivity to fosfomycin, 3 suggesting that inhibition of BSH-producing enzymes is an effective therapeutic avenue for the treatment of infections caused by fosfomycin-resistant Gram-positive organisms.…”

Purification and characterization of the Staphylococcus aureus bacillithiol transferase BstA

“…We demonstrate here that BstA also specifically utilizes bacillithiol as its thiol cofactor. In addition, studies with chelating agents showed that BstA activity is metal-dependent, as is FosB activity[14, 15]. This is an interesting observation because the only other characterized bacillithiol transferase in the DinB/YfiT-like Superfamily, B. subtilis YfiT, is likely to also be a metalloenzyme[25].…”

“…Enzymes in this Superfamily are related by structure rather than sequence based on predictions using the Superfamily database (http://supfam.org/SUPERFAMILY/). FosB, the first bacillithiol transferase described, is unrelated by structure or sequence to the YfiT-like bacillithiol transferases and is involved in detoxification of the antibiotic fosfomycin[13–15]. The YfiT-like Superfamily includes enzymes that utilize thiol cofactors glutathione, mycothiol, and bacillithiol, and the Superfamily members from the Firmicutes are predicted to be bacillithiol transferases.…”