Structural Dynamics of 15-Lipoxygenase-2 via Hydrogen-Deuterium Exchange.

Eicosanoids are inflammatory signaling lipids that are biosynthesized in response to cellular injury or threat. They were originally thought to be pro-inflammatory molecules, but members of at least one subclass, the lipoxins, are able to resolve inflammation. One step in lipoxin synthesis is the oxygenation of arachidonic acid by 15-lipoxygenase (15-LOX). 15-LOX contains two domains: a Ca2+ binding PLAT domain and a catalytic domain. 15-LOX is a soluble cytosolic protein until binding of Ca2+ to the PLAT domain promotes translocation to the membrane surface. The role of 15-LOX structural dynamics in this translocation has remained unclear. We investigated the dynamics of 15-LOX isoform B (15-LOX-2) upon binding of Ca2+ and ligands, as well as upon membrane association using hydrogen-deuterium exchange mass spectrometry (HDX-MS). We used HDX-MS to probe the solvent accessibility and backbone flexibility of 15-LOX-2, revealing significant differences in deuterium incorporation between the PLAT and catalytic domains, with the PLAT domain demonstrating higher flexibility. Comparison of HDX for 15-LOX-2 in the presence and absence of Ca2+ indicates there are few differences in structural dynamics. Furthermore, our HDX results involving nanodisc-associated 15-LOX-2 suggest that significant structural and dynamic changes in 15-LOX-2 are not required for membrane association. Our results also show that a substrate lipid binding to the active site in the catalytic domain does induce changes in incorporation of deuterium into the PLAT domain. Overall, our results challenge the previous hypothesis that Ca2+ binding induces major structural changes in the PLAT domain and support the hypothesis that is interdomain communication in 15-LOX-2.

Staphylococcus aureus CstB Is a Novel Multidomain Persulfide Dioxygenase-Sulfurtransferase Involved in Hydrogen Sulfide Detoxification.

Hydrogen sulfide (H2S) is both a lethal gas and an emerging gasotransmitter in humans, suggesting that the cellular H2S level must be tightly regulated. CstB is encoded by the cst operon of the major human pathogen Staphylococcus aureus and is under the transcriptional control of the persulfide sensor CstR and H2S. Here, we show that CstB is a multifunctional Fe(II)-containing persulfide dioxygenase (PDO), analogous to the vertebrate protein ETHE1 (ethylmalonic encephalopathy protein 1). Chromosomal deletion of ethe1 is fatal in vertebrates. In the presence of molecular oxygen (O2), hETHE1 oxidizes glutathione persulfide (GSSH) to generate sulfite and reduced glutathione. In contrast, CstB oxidizes major cellular low molecular weight (LMW) persulfide substrates from S. aureus, coenzyme A persulfide (CoASSH) and bacillithiol persulfide (BSSH), directly to generate thiosulfate (TS) and reduced thiols, thereby avoiding the cellular toxicity of sulfite. Both Cys201 in the N-terminal PDO domain (CstB(PDO)) and Cys408 in the C-terminal rhodanese domain (CstB(Rhod)) strongly enhance the TS generating activity of CstB. CstB also possesses persulfide transferase (PT; reverse rhodanese) activity, which generates TS when provided with LMW persulfides and sulfite, as well as conventional thiosulfate transferase (TST; rhodanese) activity; both of these activities require Cys408. CstB protects S. aureus against H2S toxicity, with the C201S and C408S cstB genes being unable to rescue a NaHS-induced ΔcstB growth phenotype. Induction of the cst operon by NaHS reveals that functional CstB impacts cellular TS concentrations. These data collectively suggest that CstB may have evolved to facilitate the clearance of LMW persulfides that occur upon elevation of the level of cellular H2S and hence may have an impact on bacterial viability under H2S misregulation, in concert with the other enzymes encoded by the cst operon.

X-ray crystallographic structure of BshC, a unique enzyme involved in bacillithiol biosynthesis.

Bacillithiol is produced by many Gram-positive bacteria via a pathway utilizing the enzymes BshA, BshB, and BshC. Here we report the 1.77 Å resolution crystal structure of BshC, the putative cysteine ligase in bacillithiol production. The structure reveals that BshC contains a core Rossmann fold with connecting peptide motifs (CP1 and CP2) and a unique α-helical coiled-coil domain that facilitates dimerization. The model contains citrate and glycerol in the canonical active site and ADP in a second binding pocket. The overall structure and bound ligands give insight into the function of this unique enzyme.

Structure and function of the genomically encoded fosfomycin resistance enzyme, FosB, from Staphylococcus aureus.

The Gram-positive pathogen Staphylococcus aureus is a leading cause of global morbidity and mortality. Like many multi-drug-resistant organisms, S. aureus contains antibiotic-modifying enzymes that facilitate resistance to a multitude of antimicrobial compounds. FosB is a Mn(2+)-dependent fosfomycin-inactivating enzyme found in S. aureus that catalyzes nucleophilic addition of either l-cysteine (l-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bactericidal properties. The three-dimensional X-ray crystal structure of FosB from S. aureus (FosB(Sa)) has been determined to a resolution of 1.15 Å. Cocrystallization of FosB(Sa) with either l-Cys or BSH results in a disulfide bond between the exogenous thiol and the active site Cys9 of the enzyme. An analysis of the structures suggests that a highly conserved loop region of the FosB enzymes must change conformation to bind fosfomycin. While two crystals of FosB(Sa) contain Zn(2+) in the active site, kinetic analyses of FosB(Sa) indicated that the enzyme is inhibited by Zn(2+) for l-Cys transferase activity and only marginally active for BSH transferase activity. Fosfomycin-treated disk diffusion assays involving S. aureus Newman and the USA300 JE2 methicillin-resistant S. aureus demonstrate a marked increase in the sensitivity of the organism to the antibiotic in either the BSH or FosB null strains, indicating that both are required for survival of the organism in the presence of the antibiotic. This work identifies FosB as a primary fosfomycin-modifying pathway of S. aureus and establishes the enzyme as a potential therapeutic target for increased efficacy of fosfomycin against the pathogen.

Structural and chemical aspects of resistance to the antibiotic fosfomycin conferred by FosB from Bacillus cereus.

The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M(2+)-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 (FosB(Bc)). The overall structure of FosB(Bc), at 1.27 Å 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 Ni(2+), Mn(2+), Co(2+), and Zn(2+), 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 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 L-Cys-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn(2+) 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 FosB(Bc) indicated that the enzyme has a preference for BSH over L-Cys when activated by Mn(2+) and is inhibited by Zn(2+). The fact that Zn(2+) is an inhibitor of FosB(Bc) was used to obtain a ternary complex structure of the enzyme with both fosfomycin and L-Cys bound.

Synthesis of bacillithiol and the catalytic selectivity of FosB-type fosfomycin resistance proteins.

Bacillithiol (BSH) has been prepared on the gram scale from the inexpensive starting material, D-glucosamine hydrochloride, in 11 steps and 8-9% overall yield. The BSH was used to survey the substrate and metal-ion selectivity of FosB enzymes from four Gram-positive microorganisms associated with the deactivation of the antibiotic fosfomycin. The in vitro results indicate that the preferred thiol substrate and metal ion for the FosB from Staphylococcus aureus are BSH and Ni(II), respectively. However, the metal-ion selectivity is less distinct with FosB from Bacillus subtilis, Bacillus anthracis, or Bacillus cereus.