▪ Abstract DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. In addition, these enzymes fine-tune the steady-state level of DNA supercoiling both to facilitate protein interactions with the DNA and to prevent excessive supercoiling that is deleterious. In recent years, the crystal structures of a number of topoisomerase fragments, representing nearly all the known classes of enzymes, have been solved. These structures provide remarkable insights into the mechanisms of these enzymes and complement previous conclusions based on biochemical analyses. Surprisingly, despite little or no sequence homology, both type IA and type IIA topoisomerases from prokaryotes and the type IIA enzymes from eukaryotes share structural folds that appear to reflect functional motifs within critical regions of the enzymes. The type IB enzymes are structurally distinct from a...

DNA topoisomerases: structure, function, and mechanism.

Antibiotic drug-target interactions, and their respective direct effects, are generally well characterized. By contrast, the bacterial responses to antibiotic drug treatments that contribute to cell death are not as well understood and have proven to be complex as they involve many genetic and biochemical pathways. In this Review, we discuss the multilayered effects of drug-target interactions, including the essential cellular processes that are inhibited by bactericidal antibiotics and the associated cellular response mechanisms that contribute to killing. We also discuss new insights into these mechanisms that have been revealed through the study of biological networks, and describe how these insights, together with related developments in synthetic biology, could be exploited to create new antibacterial therapies.

How antibiotics kill bacteria: from targets to networks

Summary Fluoroquinolone resistance is emerging in Gram-negative pathogens worldwide. The traditional understanding that quinolone resistance is acquired only through mutation and transmitted only vertically does not entirely account for the relative ease with which resistance develops in exquisitely susceptible organisms, or for the very strong association between resistance to quinolones and to other agents. The recent discovery of plasmid-mediated horizontally transferable genes encoding quinolone resistance might shed light on these phenomena. The Qnr proteins, capable of protecting DNA gyrase from quinolones, have homologues in water-dwelling bacteria, and seem to have been in circulation for some time, having achieved global distribution in a variety of plasmid environments and bacterial genera. AAC(6′)-Ib-cr, a variant aminoglycoside acetyltransferase capable of modifying ciprofloxacin and reducing its activity, seems to have emerged more recently, but might be even more prevalent than the Qnr proteins. Both mechanisms provide low-level quinolone resistance that facilitates the emergence of higher-level resistance in the presence of quinolones at therapeutic levels. Much remains to be understood about these genes, but their insidious promotion of substantial resistance, their horizontal spread, and their co-selection with other resistance elements indicate that a more cautious approach to quinolone use and a reconsideration of clinical breakpoints are needed.

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The worldwide emergence of plasmid-mediated quinolone resistance

The increased use of fluoroquinolones has led to increasing resistance to these antimicrobials, with rates of resistance that vary by both organism and geographic region. Resistance to fluoroquinolones typically arises as a result of alterations in the target enzymes (DNA gyrase and topoisomerase IV) and of changes in drug entry and efflux. Mutations are selected first in the more susceptible target: DNA gyrase, in gram-negative bacteria, or topoisomerase IV, in gram-positive bacteria. Additional mutations in the next most susceptible target, as well as in genes controlling drug accumulation, augment resistance further, so that the most-resistant isolates have mutations in several genes. Resistance to quinolones can also be mediated by plasmids that produce the Qnr protein, which protects the quinolone targets from inhibition. Qnr plasmids have been found in the United States, Europe, and East Asia. Although Qnr by itself produces only low-level resistance, its presence facilitates the selection of higher-level resistance mutations, thus contributing to the alarming increase in resistance to quinolones.

Mechanisms of resistance to quinolones

Elongation factor 2 kinase (eEF2k) phosphorylates and inactivates eEF2. Insulin induces dephosphorylation of eEF2 and inactivation of eEF2 kinase, and these effects are blocked by rapamycin, which inhibits the mammalian target of rapamycin, mTOR. However, the signalling mechanisms underlying these effects are unknown. Regulation of eEF2 phosphorylation and eEF2k activity is lost in cells in which phosphoinositide‐dependent kinase 1 (PDK1) has been genetically knocked out. This is not due to loss of mTOR function since phosphorylation of another target of mTOR, initiation factor 4E‐binding protein 1, is not defective. PDK1 is required for activation of members of the AGC kinase family; we show that two such kinases, p70 S6 kinase (regulated via mTOR) and p90 RSK1 (activated by Erk), phosphorylate eEF2k at a conserved serine and inhibit its activity. In response to insulin‐like growth factor 1, which activates p70 S6 kinase but not Erk, regulation of eEF2 is blocked by rapamycin. In contrast, regulation of eEF2 by stimuli that activate Erk is insensitive to rapamycin, but blocked by inhibitors of MEK/Erk signalling, consistent with the involvement of p90 RSK1 .

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Regulation of elongation factor 2 kinase by p90RSK1 and p70 S6 kinase

DNA gyrase is a type II DNA topoisomerase from bacteria that introduces supercoils into DNA1,2. It catalyses the breakage of a DNA duplex (the G segment), the passage of another segment (the T segment) through the break, and then the reunification of the break. This activity involves the opening and closing of a series of molecular ‘gates’ which is coupled to ATP hydrolysis. Here we present the crystal structure of the ‘breakage–reunion’ domain of the gyrase at 2.8 A resolution. Comparison of the structure of this 59K (relative molecular mass, 59,000) domain with that of a 92K fragment of yeast topoisomerase II (ref. 3) reveals a very different quaternary organization, and we propose that the two structures represent two principal conformations that participate in the enzymatic pathway. The gyrase structure reveals a new dimer contact with a grooved concave surface for binding the G segment and a cluster of conserved charged residues surrounding the active-site tyrosines. It also shows how breakage of the G segment can occur and, together with the topoisomerase II structure, suggests a pathway by which the T segment can be released through the second gate of the enzyme. Mutations that confer resistance to the quinolone antibacterial agents cluster at the new dimer interface, indicating how these drugs might interact with the gyrase–DNA complex.

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Crystal structure of the breakage-reunion domain of DNA gyrase

Elongation factor-2 kinase (eEF-2K) negatively regulates mRNA translation via the phosphorylation and inactivation of elongation factor-2 (eEF-2). We have shown previously that purified eEF-2K can be phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and that this induces significant Ca(2+)/calmodulin (CaM)-independent eEF-2K activity [Redpath and Proud (1993) Biochem. J. 293, 31-34]. Furthermore, elevation of cAMP levels in adipocytes also increases the level of Ca(2+)/CaM-independent eEF-2K activity to a similar extent, providing a mechanistic link between elevated cAMP and the inhibition of protein synthesis [Diggle, Redpath, Heesom and Denton (1998) Biochem. J. 336, 525-529]. Here we describe the expression of glutathione S-transferase (GST)-eEF-2K fusion protein and the identification of two serine residues that are phosphorylated by PKA in vitro. Endoproteinase Arg-C digestion of GST-eEF-2K produced two phosphopeptides that were separated by HPLC and sequenced. (32)P Radioactivity release from these peptides indicated that the sites of phosphorylation were Ser-365 and Ser-499, both of which lie C-terminal to the catalytic domain. Mutation of these sites to non-phosphorylatable residues indicated that both sites need to be phosphorylated to induce Ca(2+)/CaM-independent eEF-2K activity in vitro. However, expression of Myc-tagged eEF-2K in HEK 293 cells, followed by treatment with chlorophenylthio-cAMP (CPT-cAMP), showed that Ser-499 phosphorylation alone induced Ca(2+)/CaM-independent eEF-2K activity in cells. Co-expression of wild-type eEF-2K with luciferase resulted in a 2-3-fold reduction in luciferase expression. Expression of eEF-2K S499D resulted in a 10-fold reduction in luciferase expression despite the fact that this mutant was expressed at very low levels. This indicates that eEF-2K S499D is constitutively active when expressed in cells, thus leading to the suppression of its own expression. Our data demonstrate an important role for the phosphorylation of Ser-499 in the activation of eEF-2K by PKA and the inhibition of protein synthesis.

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Phosphorylation of elongation factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca2+/calmodulin-independent activity.

Pentaerythritol tetranitrate (PETN) reductase of Enterobacter cloacae PB2, a flavoprotein involved in the biodegradation of the explosive PETN, ethylene glycol dinitrate (EGDN) and glycerol trinitrate (GTN), was purified from an overexpressing strain of E. coli and crystallized at 293 K using the sitting-drop vapour-diffusion method. Diffraction data can be seen at 1.8 A. The primitive orthorhombic cell has a monomer in the asymmetric unit. Preliminary molecular-replacement calculations have been performed using a search model based on Old Yellow enzyme.

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Crystallization and preliminary diffraction studies of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2

Morphinone reductase from Pseudomonas putida M10, a flavoprotein involved in the degradation of morphine alkaloids, was purified from an overexpressing strain of Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.5 A. The I-centred orthorhombic cell has a monomer in the asymmetric unit. Preliminary molecular replacement calculations have been performed using Old Yellow Enzyme as the search model.

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Nita Shikotra

Papers

Crystal structure of the breakage-reunion domain of DNA gyrase

Phosphorylation of elongation factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca2+/calmodulin-independent activity.

Crystallization and preliminary diffraction studies of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2

Crystallization and preliminary diffraction studies of morphinone reductase, a flavoprotein involved in the degradation of morphine alkaloids.