Mechanism of action of the novel aminomethylcycline antibiotic omadacycline.

Omadacycline is a novel first-in-class aminomethylcycline with potent activity against important skin and pneumonia pathogens, including community-acquired methicillin-resistant Staphylococcus aureus (MRSA), ß-hemolytic streptococci, penicillin-resistant Streptococcus pneumoniae, Haemophilus influenzae, and Legionella. In this work, the mechanism of action for omadacycline was further elucidated using a variety of models. Functional assays demonstrated that omadacycline is active against strains expressing the two main forms of tetracycline resistance (efflux and ribosomal protection). Macromolecular synthesis experiments confirmed that the primary effect of omadacycline is on bacterial protein synthesis, inhibiting protein synthesis with a potency greater than that of tetracycline. Biophysical studies with isolated ribosomes confirmed that the binding site for omadacycline is similar to that for tetracycline. In addition, unlike tetracycline, omadacycline is active in vitro in the presence of the ribosomal protection protein Tet(O).

Multiple pathways of electron transfer in dimethyl sulfoxide reductase of Escherichia coli.

The catalytic subunit of dimethyl sulfoxide (Me2SO) reductase, DmsA, contains six blocks of sequence that are homologous to other members of the superfamily of prokaryotic molybdoenzymes. The amino-terminal block contains 5 conserved residues (Cys38, Cys42, Cys75, Lys28, and Arg77). Site-directed mutagenesis of these residues did not alter membrane localization but in some cases less enzyme accumulated. The activity of Me2SO reductase was monitored by measuring Me2SO-dependent anaerobic growth, benzyl viologen, or dimethylnaphthoquinol oxidase activity, and using a quinol pool-coupling assay. Only Cys75 and Lys28 mutant enzymes were able to support anaerobic growth with Me2SO suggesting a critical role for Cys38, Cys42, and Arg77. Benzyl viologen oxidase activity was retained in the mutants although with reduced efficiency in Cys42-Ser. Electron transport with dimethylnaphthoquinol was reduced in Cys38-Ser, Cys42-Ser, and Cys75-Ser and almost totally eliminated in the Arg77-Ser mutant. Cys38-Ser, Cys42-Ser, and Arg77-Ser were unable to support quinol oxidation although electron transfer from the quinol pool to the [Fe-S] centers in DmsB was normal. These results indicate that the amino-terminal region is involved in functional electron transfer from the quinol pool to Me2SO and that electrons from benzyl viologen, dimethylnaphthoquinol, and menaquinol may follow different paths within the catalytic subunit.

Consequences of removal of a molybdenum ligand (DmsA-Ser-176) of Escherichia coli dimethyl sulfoxide reductase.

We have used site-directed mutagenesis and EPR spectroscopy to examine the consequences of altering the molybdenum ligand in Escherichia coli dimethyl sulfoxide (Me2SO) reductase (DmsABC). Mutagenesis of DmsA-Ser-176 to Ala, Cys, or His abolishes both respiratory growth on Me2SO and in vitro benzyl viologen:Me2SO oxidoreductase activity. EPR spectroscopy reveals changes in the line shape and the gav of the Mo(V) signals of the S176A and S176C enzymes. The midpoint potentials (Em,7) of the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples in DmsABC are -15 and -175 mV. The Em,7 of the Mo(V)/Mo(IV) couple in the S176A mutant is 35 mV; however, the Mo(V) species could not be further oxidized with ferricyanide. Titration of the S176C mutant produced several overlapping Mo(V) species occurring at Eh > -150 mV, suggesting heterogeneity in the molybdenum environment. A Mo(V) spectrum was not visible in S176H membranes poised between -435 to 350 mV or oxidized with 200 microM ferricyanide. No differences were detected in the EPR spectra of the reduced [4Fe-4S] clusters of DmsABC and the S176A and S176H mutant enzymes; however, the S176C mutation altered the EPR line shape of one of the reduced [4Fe-4S] clusters.

Interactions between the molybdenum cofactor and iron-sulfur clusters of Escherichia coli dimethylsulfoxide reductase.

We have used site-directed mutagenesis to study the interactions between the molybdo-bis(molybdopterin guanine dinucleotide) cofactor (Mo-bisMGD) and the other prosthetic groups of Escherichia coli Me2SO reductase (DmsABC). In redox-poised preparations, there is a significant spin-spin interaction between the reduced Em,7 = -120 mV [4Fe-4S] cluster of DmsB and the Mo(V) of the Mo-bisMGD of DmsA. This interaction is significantly modified in a DmsA-C38S mutant that contains a [3Fe-4S] cluster in DmsA, suggesting that the [3Fe-4S] cluster is in close juxtaposition to the vector connecting the Mo(V) and the Em,7 = -120 mV cluster of DmsB. In a DmsA-R77S mutant, the interaction is eliminated, indicating the importance of this residue in defining the interaction pathway. In ferricyanide-oxidized glycerol-inhibited DmsAC38SBC, there is no detectable interaction between the oxidized [3Fe-4S] cluster and the Mo-bisMGD, except for a minor broadening of the Mo(V) spectrum. In a double mutant, DmsAS176ABC102SC, which contains an engineered [3Fe-4S] cluster in DmsB, no significant paramagnetic interaction is detected between the oxidized [3Fe-4S] cluster and the Mo(V). These results have important implications for (i) understanding the magnetic interactions between the Mo(V) and other paramagnetic centers and (ii) delineating the electron transfer pathway from the [4Fe-4S] clusters of DmsB to the Mo-bisMGD of DmsA.

Engineering a novel iron-sulfur cluster into the catalytic subunit of Escherichia coli dimethyl-sulfoxide reductase.

Dimethyl-sulfoxide reductase (DmsABC) is a complex [Fe-S] molybdoenzyme that contains four [4Fe-4S] clusters visible by electron paramagnetic resonance (EPR) spectroscopy. The enzyme contains four ferredoxin-like Cys groups in the electron transfer subunit, DmsB, and an additional group of Cys residues in the catalytic subunit, DmsA. Mutagenesis of the second Cys, Cys-38, in the DmsA group to either Ser or Ala promotes assembly of a fifth [Fe-S] cluster into the mutant enzyme. The EPR spectra, the temperature dependences, and the microwave power dependences demonstrate that the new clusters are [3Fe-4S] clusters. The [3Fe-4S] clusters in both of the C38S and C38A mutant enzymes are relatively unstable in redox titrations and have midpoint potentials of approximately 178 and 140 mV. Mutagenesis of the DmsA Cys group to resemble a sequence capable of binding an [4Fe-4S] cluster did not change the cluster type but reduced the amount of the cluster present in this mutant enzyme. This report demonstrates that all four EPR detectable [Fe-S] clusters in the wild-type enzyme are ligated by DmsB. Wild-type DmsA does not ligate an [Fe-S] cluster that is visible by EPR spectroscopy.

The topology of the anchor subunit of dimethyl sulfoxide reductase of Escherichia coli.

The terminal electron transfer enzyme Me2SO reductase of Escherichia coli is a heterotrimeric enzyme composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic polytopic anchor subunit (DmsC). The topology of DmsC has been studied using phoA (alkaline phosphatase) and blaM (beta-lactamase) gene fusions. The results of analyzing the properties of proteins produced by the fusions suggests a structure with eight transmembrane helices. Both the amino and carboxyl termini are exposed to the periplasm. The entire DmsC polypeptide is necessary to anchor DmsAB to the membrane as fusions with truncated DmsC were not functional and soluble DmsAB accumulated in the cytoplasm. A dmsC-phoA fusion in the termination codon of dmsC generated a chimeric enzyme with functional Me2SO reductase and alkaline phosphatase activity. Quantitation of the minimal inhibitory concentration of ampicillin for the dmsC-blaM fusions indicated that different transmembrane helices had differing signal sequence activity.

Host mutations (miaA and rpsL) reduce tetracycline resistance mediated by Tet(O) and Tet(M).

The effects of mutations in host genes on tetracycline resistance mediated by the Tet(O) and Tet(M) ribosomal protection proteins, which originated in Campylobacter spp. and Streptococcus spp., respectively, were investigated by using mutants of Salmonella typhimurium and Escherichia coli. The miaA, miaB, and miaAB double mutants of S. typhimurium specify enzymes for tRNA modification at the adenosine at position 37, adjacent to the anticodon in tRNA. In S. typhimurium, this involves biosynthesis of N6-(4-hydroxyisopentenyl)-2-methylthio-adenosine (ms2io6A). The miaA mutation reduced the level of tetracycline resistance mediated by both Tet(O) and Tet(M), but the latter showed a greater effect, which was ascribed to the isopentenyl (i6) group or to a combination of the methylthioadenosine (ms2) and i6 groups but not to the ms2 group alone (specified by miaB). In addition, mutations in E. coli rpsL genes, generating both streptomycin-resistant and streptomycin-dependent strains, were also shown to reduce the level of tetracycline resistance mediated by Tet(O) and Tet(M). The single-site amino acid substitutions present in the rpsL mutations were pleiotropic in their effects on tetracycline MICs. These mutants affect translational accuracy and kinetics and suggest that Tet(O) and Tet(M) binding to the ribosome may be reduced or slowed in the E. coli rpsL mutants in which the S12 protein is altered. Data from both the miaA and rpsL mutant studies indicate a possible link between stability of the aminoacyl-tRNA in the ribosomal acceptor site and tetracycline resistance mediated by the ribosomal protection proteins.

Ribosomal protection from tetracycline mediated by Tet(O): Tet(O) interaction with ribosomes is GTP-dependent.

Tet(O) mediates tetracycline resistance by protecting the ribosome from inhibition. A recombinant Tet(O) protein with a histidine tag was purified and its activity in protein synthesis characterized. Tetracycline inhibited the rate of poly(Phe) synthesis, producing short peptide chains. Tet(O)-His was able to restore the elongation rate and processivity. 70S ribosomes bound tetracycline with high affinity. Tet(O)-His in the presence of GTP, but not GDP or GMP, reduced the affinity of the ribosomes for tetracycline. Non-hydrolyzable GTP analogs in the presence of the factor were also able to interfere with tetracycline binding. Ribosomes increased the affinity of Tet(O)-His for GTPgammaS. Tet(O), 70S ribosomes and GTPgammaS formed a complex that could be isolated by gel filtration. The GTP conformer is the active form of Tet(O) that interacts with the ribosome. GTP binding is necessary for Tet(O) activity.

Localization of the ribosomal protection protein Tet(O) on the ribosome and the mechanism of tetracycline resistance.

Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance. It is a G protein that shows significant sequence similarity to elongation factor EF-G. Here we present a cryo-electron microscopic reconstruction, at 16 A resolution, of its complex with the E. coli 70S ribosome. Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site. Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G. However, there are important differences. One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site. Our findings give insights into the mechanism of tetracycline resistance.