A novel hydroperoxo-copper(II) complex with a square-planar geometry has been prepared, which has exhibited a higher selectivity and catalytic reactivity for dimethyl sulfide, in contrast to that with a trigonal-bipyramidal one.

Subunit A and B proteins of DNA gyrase were separately purified from fluoroquinolone-resistant Escherichia coli GN14181 (MIC of ofloxacin, 100 gLg/ml) and susceptible strain KL-16. The supercoiling activities of reconstituted Ar+Br (r, resistant) and Ar+Bs (s, susceptible) were 250-fold more resistant to new fluoroquinolones thsn those of As+Bs and As+Br.DNA gyrase was discovered by Geilert et al. (8) as an activity in Escherichia coli that catalyzes the ATP-coupled negative supercoiling of DNA. In addition to supercoiling activity, the enzyme causes the relaxation of supercoiling DNA in the absence of ATP, and the binding of the enzyme to DNA leads to double-strand breakage at specific sites promoted by quinolones and sodium dodecyl sulfate (4,5,10,17,20). The enzyme activities result from breakage and rejoining of both strands of duplex DNA (3,5). The active gyrase holoenzyme is a tetramer composed of two subunits, A2 and B2 (10, 15). It is believed that subunit A protein is the target of quinolones because nalidixic acid-resistant gyrA mutants are also resistant to quinolones (11,12). New fluoroquinolones, such as ofloxacin, norfloxacin, ciprofloxacin, and enoxacin, have broad spectra against gramnegative and -positive bacteria (13,14,16,19), and they strongly inhibit the supercoiling activity of DNA gyrase from E. coli . However, there have been no reports on the inhibitory properties of DNA gyrase from clinical isolates.In this report, we present the purification and properties of subunit A and B proteins of DNA gyrase from a fluoroquinolone-resistant isolate, E. coli GN14181.E. coli GN14181 was isolated from an upper urinary tract infection and identified by the method ofBergey's Manual of Systematic Bacteriology (3). E. coli K-12 strains KL-16 (Hfr, thi, relA) and MH-5, its gyrAr derivative, were also used (2, 9).We received the following compounds as gifts from their manufacturers: ofloxacin (OFX), norfloxacin (NFX), ciprofloxacin (CPX), enoxacin (ENX), pipemidic acid, and nalidixic acid (NA). Novobiocin (NB) and coumermycin Al (COU) were purchased from Sigma Chemical Co. purified by using a heparin-Sepharose CL-6B (Pharmacia Fine Chemicals) column. Active fractions, which eluted around 0.3 M NaCl, were pooled after dialysis against TGED buffer. The B subunit was eluted with 5 M urea from an NB-Sepharose column.The assay of supercoiling activity was modified from previous reports (6, 7). One unit of enzyme activity was defined as the amount that brought 50% of relaxed pBR322 to the supercoiled position in agarose gel electrophoresis as described by Gellert et al. (8). The reaction mixture containing subunit A and B proteins (each, 0.1 ,ug of protein), drug solution, and pBR322 relaxed by topoisomerase I (Bethesda Research Laboratories, Inc.) was incubated at 37°C for 1 h, and the reaction was stopped by the addition of proteinase K (20 ,ug/ml). The mixtures were subjected to 0.8% agarose gel electrophoresis. The gel was stained with ethidium bromide (0.5 ,ug/ml) and photographed by using UV light. The ne...

Subunits A and B of DNA gyrase were purified from Pseudomonas aeruginosa PAO1 and its mutant, which was resistant to nalidixic acid. Inhibition tests of DNA gyrases reconstituted with a combination of subunits from the two strains showed that an alteration of subunit A but not subunit B caused bacteria to resist fluoroquinolones.New fluoroquinolones, such as norfloxacin (NFX), ofloxacin (OFX), enoxacin, and ciprofloxacin (CPX) show activity against a broad spectrum of gram-negative and gram-positive bacteria, including nalidixic acid (NA)-resistant strains (11,20,25). From studies of in vitro DNA replication systems, these quinolones appear to inhibit DNA syhthesis by inhibiting the activity of DNA gyrase (10, 24). DNA gyrase, which catalyzes the supercoiling of relaxed closed circular DNA by coupling with the hydrolysis of ATP, was found in Escherichia coli (5) and was considered to be an essential enzyme for the DNA replication of chromosomes, plasmids, and bacteriophage (12,13,23). DNA gyrase from E. coli consists of two subunits, A and B (3, 7). Subunit A is a dimer of a 105,000-dalton protein encoded by the gyrA gene, which determines resistance to NA, and subunit B consists of a 95,000-dalton protein encoded by the gyrB gene, which determines resistance to novobiocin and coumermycin Al (4,7,9,19,29).Strains of Pseudomonas aeruginosa are the most important pathogens, because they are often isolated from clinical specimens and are resistant to various antibacterial agents (2). Benbrook and Miller (1) reported that NFX inhibits both the replication of DNA in growing cells of P. aeruginosa and the supercoiling activity of DNA gyrase in vitro. Further, we reported that OFX and CPX, in addition to NFX, strongly inhibit the supercoiling activity of DNA gyrase from P. aeruginosa PAO1 (24).It was reported that the NA resistance mechanisms of P. aeruginosa were chromosomal mutations of nalA or nalB genes (22). The nalA gene is located at approximately 51 min, and the nalB gene is located at approximately 31 min on the genetic map of P. aeruginosa PAO (22), although enzymatic studies have not been done until recently.In this study, we describe the purification procedure and show some properties of subunit A and subunit B proteins of DNA gyrase from a mutant (Nalr), compared with those of the parent strain P. aeruginosa PAO1, to clarify resistance mechanisms of P. aeruginosa to quinolones. P. aeruginosa PAO1 (8) We obtained the following compounds as gifts from the manufacturers: NFX, OFX, enoxacin, CPX, pipemidic acid (PPA), and NA. To determine the MICs of drugs, the agar dilution method was employed. One loopful (ca. 3 x 104 CFU) of a diluted culture was inoculated onto modified Mueller-Hinton agar (Nissui Seiyaku Co., Ltd.) containing serial twofold dilutions of a drug. The MICs were determined after incubation at 37°C for 18 h.P. aeruginosa was grown in 2 liters of medium B (18) and harvested at late log phase. Cells were washed once with 300 ml of saline solution and suspended in an equal volume of 10 mM Tris ...

A mononuclear copper(II)-hydroperoxo species has been generated by the reaction of Cu(I)-H2BPPA complex with dioxygen, which illustrates the enzymatic reaction process of the CuB site in the DbetaM and PHM.

Radical halo-nitration of alkenes using iron(III) nitrate nonahydrate and halogen salt has been developed. The present reaction proceeds by radical addition of nitrogen dioxide generated by thermal decomposition of iron(III) nitrate nonahydrate and subsequent trapping of the resultant radical by a halogen atom in the presence of halogen salt. Application of this method to synthesis of nitroalkenes is also described. The practicality of the present method using nontoxic and inexpensive iron reagents has been shown by the application to broad alkenes.