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Kid and Kis are, respectively, the toxin and antitoxin encoded by the parD operon of plasmid R1. The recently solved crystal structure of Kid has revealed that this protein closely resembles the CcdB toxin of plasmid F. In CcdB, the residues involved in toxicity are located at the carboxy-terminal end of the protein. However, an analogous information on the Kid toxin was not available. Here, we have characterized a collection of non-toxic mutants of the Kid protein and identified the residues that affected the toxicity but not the co-regulatory activity of Kid. These are located in two discrete regions of the protein, at the amino and carboxy-terminal ends. Particularly, residues E18 and R85, that are conserved in the Escherichia coli ChpAK and RelE toxins, are affected by amino-acid changes that alter neither the overall structure of the protein nor its state of association, as shown by CD and sedimentation equilibrium analyses. However, thermal denaturation and intrinsic tryptophan fluorescence emission data point to subtle local changes at the N-terminal end of the protein. The implications of these results in the current model on the structure and function of Kid-related bacterial toxins are discussed.

Enoyl acyl carrier protein (ACP) reductase catalyses the last reductive step of fatty acid biosynthesis, reducing the enoyl group of a growing fatty acid chain attached to ACP to its acyl product using NAD(P)H as the cofactor. This enzyme is the target for the diazaborine class of antibacterial agents, the biocide triclosan, and one of the targets for the front-line anti-tuberculosis drug isoniazid. The structures of complexes of Escherichia coli enoyl-ACP reductase (ENR) from crystals grown in the presence of NAD+ and a family of diazaborine compounds have been determined. Analysis of the structures has revealed that a mobile loop in the structure of the binary complex with NAD+ becomes ordered on binding diazaborine/NAD+ but displays a different conformation in the two subunits of the asymmetric unit. The work presented here reveals how, for one of the ordered conformations adopted by the mobile loop, the mode of diazaborine binding correlates well with the activity profiles of the diazaborine family. Additionally, diazaborine binding provides insights into the pocket on the enzyme surface occupied by the growing fatty acid chain.

Enoyl acyl carrier protein reductase catalyses the last reductive step of fatty acid biosynthesis, reducing an enoyl acyl carrier protein to an acyl-acyl carrier protein with NAD(P)H as the cofactor. The crystal structure of enoyl reductase (ENR) from Escherichia coli has been determined to 2.1 A resolution using a combination of molecular replacement and isomorphous replacement and refined using data from 10 A to 2.1 A to an R-factor of 0.16. The final model consists of the four subunits of the tetramer, wherein each subunit is composed of 247 of the expected 262 residues, and a NAD+ cofactor for each subunit of the tetramer contained in the asymmetric unit plus a total of 327 solvent molecules. There are ten disordered residues per subunit which form a loop near the nucleotide binding site which may become ordered upon substrate binding. Each monomer is composed of a seven-stranded parallel beta-sheet flanked on each side by three alpha-helices with a further helix lying at the C terminus of the beta-sheet. This fold is highly reminiscent of the Rossmann fold, found in many NAD(P)H-dependent enzymes. Analysis of the sequence and structure of ENR and comparisons with the family of short-chain alcohol dehydrogenases, identify a conserved tyrosine and lysine residue as important for catalytic activity. Modelling studies suggest that a region of the protein surface that contains a number of strongly conserved hydrophobic residues and lies adjacent to the nicotinamide ring, forms the binding site for the fatty acid substrate., (Copyright 1998 Academic Press)

The NADP-dependent beta-keto acyl carrier protein reductase (BKR) from E. coli has been crystallized by the hanging-drop method of vapour diffusion using poly(ethylene glycol) of average molecular weight 1450. The crystals belong to the hexagonal space group P6122 or P6522 with unit-cell dimensions a = b = 67.8, c = 355.8 A. Calculated values for Vm and consideration of the packing suggest that the asymmetric unit contains a dimer. BKR catalyses the first reductive step in the elongation cycle of fatty-acid biosynthesis. It shares extensive sequence homology with the enzyme which catalyzes the second reductive step in the cycle, enoyl acyl carrier protein reductase (ENR), and thus provides an opportunity to study the evolution of enzyme function in a metabolic pathway. The structure determination will permit the analysis of the molecular basis of its catalytic mechanism and substrate specificity.

Comparison of the structure of Escherichia coli RuvA with other proteins in the Protein Data Bank gives insights into the probable modes of association of RuvA with the Holliday junction during homologous recombination. All three domains of the RuvA protein possess striking structural similarities to other DNA-binding proteins. Additionally, the second domain of RuvA contains two copies of the helix-hairpin-helix (HhH) structural motif, which has been implicated in non-sequence-specific DNA binding. The two copies of the motif are related by approximate 2-fold symmetry and may form a bidentate DNA-binding module. The results described provide support for the organization of the arms of the DNA in our RuvA/Holliday junction complex model and support the involvement of the HhH motifs in DNA binding., (Copyright 1998 Academic Press Limited.)

Here we confirm and extend our previous studies demonstrating that the mutagenic potency of 1,2-dibromoethane (DBE) and dibromomethane (DBM) is markedly enhanced (not prevented) in bacteria expressing the O6-alkylguanine-DNA alkyltransferase (ATase) encoded by the Escherichia coli ogt gene. We demonstrate that, in close parallel with mutagenesis, the Ogt ATase sensitizes the bacteria to the lethal effects of these carcinogens, suggesting that one or more of the potentially mutagenic lesions induced by DBE and DBM in the presence of Ogt has additional lethal capacity. We further demonstrate that the sensitization to both lethality and mutagenesis by DBE and DBM is a property shared by other DNA alkyltransferases. This objective was accomplished by quantifying the induction of mutations and lethal events in ogt- ada- E. coli expressing an exogenous bacterial or mammalian ATase from a multicopy plasmid. Mammalian recombinant ATases enhanced the lethal and mutagenic actions of DBE and suppressed the lack of sensitivity of the vector-transformed bacteria to DBM. In most cases the order of effectiveness of the ATases ranked: murine > human > Ogt > rat. Further comparisons included the full-length Ada ATase from E. coli and a truncated Ada version (T-ada) that retains the O6-methylguanine binding domain of the protein. The full-length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by DBE and DBM. The T-ada ATase provided less sensitization than Ada to lethality by DBE, but of the three bacterial ATases T-ada yielded the highest sensitization to mutagenesis by this compound. T-ada and Ada ATases were in general less effective than the mammalian versions, with the exception of the rat recombinant ATase. The effectiveness of the different mammalian and bacterial ATases in promoting the deleterious actions of dibromoalkanes was compared with the effectiveness of these proteins in suppressing the lethal and mutagenic effects induced by N-nitroso-N-methylurea. The ability to sensitize E. coli to the lethal and mutagenic effects of DBE and DBM seems restricted to DNA alkyltransferase, since overexpression of thioredoxin (Trx) or glutaredoxin (Grx1) in ogt- ada- cells showed no effect, in spite of the reported potential of bioactive dihaloethane-derived species to alkylate Trx.

We investigated the relative contribution of the two Escherichia coli DNA alkyltransferases (ATases) to the increased sensitivity of ATase-deficient bacteria to the mutagenic and lethal effects of chloroethylnitrosoureas (CNU). The ogtencoded protein was the principal determinant in resistance to the mutagenic effects of CNU in E.coli. Thus, only when the ogt gene was inactivated was sensitivity to mutagenesis greatly increased; the contribution of inactivation of the ada gene was relatively minor. Furthermore, induction of the adaptive response provided essentially no protection against CNU mutagenesis in either an ogt+ or ogt- background. Finally, overexpression of the ogt gene into ogt- ada- double mutants provided the greatest protection against CNU; introduction of the full-length or truncated ada gene was protective, but to a much lesser extent. Mammalian ATases were not as protective against mutation induction by CNU as Ogt, even though they were apparently expressed at higher level. In order of effectiveness the ATases ranked Ogt > human > truncated Ada = Ada > rat. This order was not observed in the protection against killing by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, where truncated Ada = human > Ogt > rat = Ada. Higher mutation frequency and toxicity were observed in uvr- mutants, suggesting that one or more of the potentially mutagenic and/or toxic lesions are also substrates for the excision repair proteins.

We have previously reported the isolation of an Escherichia coli K12 mutant that is extremely sensitive to mutagenesis by low doses of ethylating agents. We now show by Southern analysis that the mutation involves a gross deletion covering at least the ogt and fnr genes and that no O6-alkylguanine-DNA-alkyltransferase activity is present in cell-free extracts of an ada::Tn10 derivative of these bacteria. Confirmation that sensitisation to ethylation-induced mutagenesis was attributable to ogt and not to any other loci covered by the deletion was obtained by constructing derivatives. Thus an ogt::kanr disruption mutation was introduced into the parental ogt+ bacteria, and the ogt::kanr mutation was then eliminated by cotransduction of ogt+ with the closely linked Tetr marker (zcj::Tn10). The delta(ogt-fnr) deletion or ogt::kanr disruption mutants were highly sensitive to ethyl methanesulphonate-induced mutagenesis, as measured by the induction of forward mutations to L-arabinose resistance (Arar). Furthermore, the number of Arar mutants increased linearly with dose, unlike the case in ogt+ bacteria, which had a threshold dose below which no mutants accumulated. Differences in mutability were even greater with propyl methanesulphonate. Overproduction of the ogt alkyltransferase from a multicopy plasmid reduced ethylmethanesulphonate-induced mutagenesis in the ogt- mutant strains and also methylmethanesulphonate mutagenesis in ada- bacteria. A sample of AB1157 obtained from the E. coli K12 genetic stock centre also had a deletion covering the ogt and fnr genes. Since such deletions greatly influence the mutagenic responses to alkylating agents, a survey of the presence of the ogt gene in the E. coli K12 strain being used is advisable.

The action of O6-benzylguanine (O6-BzlG) on recombinant mammalian and Escherichia coli O6-alkylguanine-DNA alkyltransferases (ATase; EC 2.1.1.63; methylated-DNA-protein-cysteine methyltransferase) was compared by preincubation of these proteins with the base, followed by measurement of residual ATase activity using [3H]methylated substrate DNA. All of the mammalian proteins examined were inactivated by O6-BzlG (Chinese hamster: I40, 0.04 microM; human and rat: I40, 0.06 microM); however, the murine ATase was substantially more resistant requiring 4-5 fold higher concentrations of O6-BzlG to achieve the same levels of inactivation (I40, 0.28 microM). A similar differential inactivation was seen with human and murine ATases when extracts of 3T6 (murine) cells and Raji (human) cells were compared. Of the two E. coli ATase proteins, only the ogt-encoded protein was inactivated, but approximately 400 times more O6-BzlG was required to achieve a level of inactivation similar to that seen with the human protein (I40, 24.8 microM). When O6-BzlG was present in an oligonucleotide, the differential effect on the murine, human and ogt-encoded ATases was not seen and only the ada-encoded ATase remained refractory under the conditions used.

The three-dimensional crystal structure of met repressor, in the presence or absence of bound corepressor (S-adenosylmethionine), shows a dimer of intertwined monomers, which do not have the helix-turn-helix motif characteristic of other bacterial repressor and activator structures. We propose that the interaction of met repressor with DNA occurs through either a pair of symmetry-related alpha-helices or a pair of beta-strands, and suggest a model for binding of several dimers to met operator regions.

The met repressor from Escherichia coli has been crystallized in space group P21, with unit cell dimensions a = 35.6 A, b = 62.6 A, c = 44.5 A, beta = 102.4 degrees and one aporepressor dimer per asymmetric unit. Preliminary X-ray diffraction photographs show measurable intensities to beyond 1.5 A resolution, and the crystal form is ideally suited to high-resolution crystallographic analysis (1 A = 0.1 nm).

We present biochemical and genetic data to support the hypothesis that the Escherichia coli met repressor, MetJ, binds to synthetic and natural operator sequences in tandem arrays such that repression depends not only on the affinity of the DNA-protein interaction, but also on protein-protein contacts along the tandem array. This represents a novel form of regulatory switch. Furthermore, there seems to be homology between the organization of the met and trp operators.