FtsZ is an essential bacterial guanosine triphosphatase and homolog of mammalian beta-tubulin that polymerizes and assembles into a ring to initiate cell division. We have created a class of small synthetic antibacterials, exemplified by PC190723, which inhibits FtsZ and prevents cell division. PC190723 has potent and selective in vitro bactericidal activity against staphylococci, including methicillin- and multi-drug-resistant Staphylococcus aureus. The putative inhibitor-binding site of PC190723 was mapped to a region of FtsZ that is analogous to the Taxol-binding site of tubulin. PC190723 was efficacious in an in vivo model of infection, curing mice infected with a lethal dose of S. aureus. The data validate FtsZ as a target for antibacterial intervention and identify PC190723 as suitable for optimization into a new anti-staphylococcal therapy.

A comparison of L. plantarum and T. thermophilus catalase structures reveals the existence of two distinct structural classes, differing in monomer design and the organization of their active sites, within the manganese catalase family. These differences have important implications for catalysis and may reflect distinct biological functions for the two enzymes, with the L. plantarum enzyme serving as a catalase, while the T. thermophilus enzyme may function as a catalase/peroxidase.

A general approach for simulation of EPR spectra of mixed-valence dimanganese complexes and proteins is presented, based on the theory of Sage et al. (J. Am. Chem. Soc. 1989, 111, 7239-7247), which overcomes limitations inherent in the theory of strongly coupled ions. This enables explanation of "anomalous" spectral parameters and extraction of accurate g tensors and 55Mn magnetic hyperfine tensors from which the spatial distribution of the unpaired spin density, the electronic configuration, and ligand field parameters have been obtained. This is used to analyze highly accurate simulations of the EPR spectra, obtained by least-squares fits of two mixed valence oxidation states, from a series of dimanganese(II,III) and dimanganese(III,IV) complexes and from the dimanganese catalase enzyme, MnCat(II,III) and MnCat(III,I V), from Thermus thermophilus. The sign of the 55Mn dipolar hyperfine anisotropy ( ) reveals that the valence orbital configuration of the Mn(III) ion in MnCat(III,IV) and all dimanganese(III,IV) complexes possessing sterically unconstrained bis(^-oxo) bridges is dT* 123(dz2)1, with the antibonding dz: electron oriented perpendicular to the plane of the 2(µ-0)2 rhombus. This accounts for the strong Mn-0 bonding and slow ligand exchange kinetics widely observed. The asymmetry of the spin density of Mn(III) increases substantially from / = 0.27 in MnCat(III,I V) to 0.46 in MnCat(II,III), reflecting a change in manganese coordination. Comparison with model complexes suggest this may be due to protonation and opening of the (µ-0)2 bridge upon reduction to yield a single µbridge. The presence of strong Mn-O bonding in an unreactive (µ-0)2 core of MnCat(III,IV) offers a plausible explanation for the 1012 slower kinetics of peroxide dismutation compared to what is observed for the physiologically important oxidation state MnCat(II,II). For the dimanganese(II,III) oxidation state, the theory also provides the first explanation for the anomalously large (~30%) 55Mn(II) hyperfine anisotropy in terms of admixing of the S' = 3/2 excited state into the ground state (S = '/2) via the zero-field splitting interaction of Mn(III). This "transferred" anisotropy obscures the otherwise typical isotropic high-spin 3d5 orbital configuration of Mn(II). An estimate of the ratio of the zero-field splitting to the Heisenberg exchange interaction (D/J) is obtained. The theory also explains the unusual 12-line EPR spectrum for a weakly coupled dimanganese(III,IV) complex (Larson et al. J. Am. Chem. Soc. 1992,114,6263-6265), in contrast to the typical 16-line "multiline" spectra seen in strongly coupled dimanganese(III,IV) complexes. The theory shows this is due to a weak J = -10 cm-1 which results in a D/J ratio approaching unity and not to unusual intrinsic magnetic hyperfine parameters of the Mn ions.

Peptide deformylase (PDF) is an essential bacterial metalloenzyme which deformylates the N-formylmethionine of newly synthesized polypeptides and as such represents a novel target for antibacterial chemotherapy. To identify novel PDF inhibitors, we screened a metalloenzyme inhibitor library and identified an N-formylhydroxylamine derivative, BB-3497, and a related natural hydroxamic acid antibiotic, actinonin, as potent and selective inhibitors of PDF. To elucidate the interactions that contribute to the binding affinity of these inhibitors, we determined the crystal structures of BB-3497 and actinonin bound to Escherichia coli PDF at resolutions of 2.1 and 1.75 Å, respectively. In both complexes, the active-site metal atom was pentacoordinated by the side chains of Cys 90, His 132, and His 136 and the two oxygen atoms of N-formyl-hydroxylamine or hydroxamate. BB-3497 had activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis, and activity against some gram-negative bacteria. Time-kill analysis showed that the mode of action of BB-3497 was primarily bacteriostatic. The mechanism of resistance was via mutations within the formyltransferase gene, as previously described for actinonin. While actinonin and its derivatives have not been used clinically because of their poor pharmacokinetic properties, BB-3497 was shown to be orally bioavailable. A single oral dose of BB-3497 given 1 h after intraperitoneal injection of S. aureus Smith or methicillin-resistant S. aureus protected mice from infection with median effective doses of 8 and 14 mg/kg of body weight, respectively. These data validate PDF as a novel target for the design of a new generation of antibacterial agents.Ribosome-mediated synthesis of proteins starts with a methionine residue. In prokaryotes, the amino group of the methionyl moiety carried by the initiator tRNA fMet is N formylated by formyltransferase prior to its incorporation into a polypeptide. Consequently, N-formylmethionine is always present at the N terminus of a nascent bacterial polypeptide. However, most mature proteins do not retain the N-formyl group or the terminal methionine residue. Following translation, the formyl group is hydrolyzed by peptide deformylase (PDF), which is necessary for further processing at the N terminus by methionine aminopeptidase (32). Deformylation is therefore a crucial step in bacterial protein biosynthesis, and PDF is essential for bacterial growth (23). The gene encoding PDF (def) is present in all sequenced pathogenic bacterial genomes and has no mammalian counterpart, making it an attractive target for antibacterial chemotherapy. Although the enzyme has been known for 30 years, it has proved difficult to isolate and characterize due to its apparent instability. Recently, two X-ray crystal structures and a solution structure of PDF have been determined (5, 9, 12), identifying PDF as a new class of metalloenzyme related in structure to the metalloproteinase superfamily. ...

The dimanganese (II,II) catalase from Thermus thermophilus, MnCat(II,II), arginase from rat liver, Arg(II,II), and several dimanganese(II,II) compounds, LMn2XY2, which are functional catalase mimics, all possess a pair of coupled Mn(II) ions in their catalytic sites. For each of these, we have measured by EPR spectroscopy the relative energies separating the three lowest electronic states (singlet, triplet, and quintet), described a general method for extracting the individual spectra for these states by multicomponent analysis, and determined the Mn-Mn separation. The triplet-singlet and quintet-singlet energy gaps were modeled well by fitting the temperature dependence of the EPR intensities to a Boltzmann expression for a pair of Mn(II) ions coupled by isotropic Heisenberg spin exchange (-2JS1S2). This dependence indicates diamagnetic ground states with delta E10 (cm-1) = magnitude of 2J = 4 and 11.2 cm-1 for Arg-(II,II)(+borate) and MnCat(II,II)(phosphate), respectively. This large difference in magnitude of 2J reflects either a difference in the bridging ligands or, possibly, a weaker ligand field (larger ionization potential) for the Mn(II) ions in arginase. In n-butanol/CH2Cl2 the triplet-singlet energy gaps for [LMn2(CH3CO2)](C1O4)2 (1), [LMn2(CH3CO2)3] (2), and [LMn2Cl3] (3), where HL = N,N,N',N'-tetrakis(2-methylenebenzimidazole)-1,3-diaminopropan+ ++-2-ol, are 23-24 cm-1. Comparison of the Heisenberg exchange interaction constants for more than 30 dimanganese(II,II) complexes suggests a possible bridging structure of (mu-OH)(mu-carboxylate)1-2 for MnCat(II,II), while the 3-fold weaker coupling in Arg(II,II) suggests mu-aqua in place of mu-hydroxide. EPR spectra of both the triplet and quintet electronic states were extracted and found to exhibit zero-field splittings (ZFS) and resolved 55Mn hyperfine splittings indicating spin-coupled Mn2-(II,II) species. The major ZFS interaction could be attributed to the magnetic dipole-dipole interaction between the Mn(II) ions. A linear correlation is observed between the crystallographically determined Mn-Mn distance and the ZFS of the quintet state (D2) for five dimanganese pairs for which both data sets are available. Using this correlation, the Mn-Mn distance in Arg(II,II) is predicted to be 3.36-3.57 A for the native enzyme (multiple forms) and 3.59 A for MnCat(II,II)(phosphate). Addition of the inhibitor borate to Arg(II,II) simplifies the ZFS, indicative of conversion to a single species with mean Mn-Mn separation of 3.50 A. The second metal ion in dinuclear complexes possessing a shared bridging ligand has been shown to attenuate the strength of the mu-ligand field potential, as monitored by the strength of the single ion ZFS.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

The superoxidized Mn III Mn IV state of dimanganese catalase from Thermus thermophilus and a series of structurally similar Mn III Mn IV complexes were investigated using continuous wave (CW) and pulsed EPR spectroscopy at X-(9 GHz), Q-(34 GHz), and W-band (94 GHz) frequencies. The bis(µ-oxo) or bis(µ-oxo)-(µ-carboxylato) bridged complexes exhibit strong antiferromagnetic coupling (|J| > 100 cm -1 ). Relevant EPR parameters (G-and 55 Mn hyperfine coupling tensors) are obtained by spectral simulations and yield a consistent data set for all frequency bands. Two mechanisms that lead to EPR line broadening are discussed. The advantage of our fitting strategy, i.e., precise determination of the G-tensor from high-field/frequency and hyperfine couplings from low-frequency EPR spectra, over conventional procedures is outlined. Comparison of the G-and 55 Mn hyperfine tensors of the model complexes with those of dimanganese catalase show that both values are sensitive probes for small structural changes.

The bacterioferritin (BFR) of Escherichia coli is a heme-containing iron storage molecule. It is composed of 24 identical subunits, which form a roughly spherical protein shell surrounding a central iron storage cavity. Each of the 12 heme moieties of BFR possesses bis-methionine axial ligation, a heme coordination scheme so far only found in bacterioferritins. Members of the BFR family contain three partially conserved methionine residues (excluding the initiating methionine) and in this study each was substituted by leucine and/or histidine. The Met 52 variants were devoid of heme, whereas the Met 31 and Met 86 variants possessed full heme complements and were spectroscopically indistinguishable from wild-type BFR. The heme-free Met 52 variants appeared to be correctly assembled and were capable of accumulating iron both in vivo and in vitro. No major differences were observed in the overall rate of iron accumulation for BFR-M52H, BFR-M52L, and the wildtype protein. The iron contents of the Met 52 variants, as isolated, were at least 4 times greater than for wild-type BFR. This study is consistent with the reported location of the BFR heme site at the 2-fold axis and shows that heme is unnecessary for BFR assembly and iron uptake.