Genetic and molecular organization of the alkylbenzene catabolism operon in the psychrotrophic strain Pseudomonas putida 01G3.
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The 11-kb sequence encompassing the alkylbenzene upper pathway in Pseudomonas putida 01G3, a psychrotrophic strain able to degrade alkylbenzenes at low temperatures, was characterized. Together with a potential regulator (EbdR), six putative enzymes (EbdAaAbAcAdBC) were identified, and they exhibited highly significant similarities with enzymes implicated in the equivalent pathway in P. putida RE204. ebd genes appeared to be preferentially induced by ethylbenzene. Multiple-alignment data and growth rate measurements led us to classify 01G3 and closely related strains in two groups with distinct substrate specificities. Close to identified genes, remnants of IS5-like elements provided insight into the evolution of catabolic sequences through rearrangements from a less complex ancestral cluster. (+info)
Genetic and physiological characterization of ohr, encoding a protein involved in organic hydroperoxide resistance in Pseudomonas aeruginosa.
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The ohr (organic hydroperoxide resistance) gene product of Pseudomonas aeruginosa was essential for optimal resistance to organic hydroperoxides (OHPs) but not to hydrogen peroxide or paraquat. A Deltaohr mutant was hypersusceptible to OHPs in disk inhibition assays and showed enhanced killing by OHPs in liquid culture. The ohr gene product was demonstrated to contribute to the decomposition of OHPs. Transcription of ohr was induced up to 15-fold upon exposure to OHPs, and this induction was independent of OxyR. Somewhat enhanced ohr-lacZ activity was detected in mutant strains affected in ohr, ahpC, and oxyR, and this phenotype correlated with hypersusceptibility to OHPs, suggesting overlapping or compensatory functions of the ohr and ahpC gene products. A single transcriptional start site for ohr was determined, and ohr transcripts were abundant in cells treated with a sublethal dose of OHPs but not in cells treated with paraquat. An 84-bp portion upstream of the ohr mRNA start site was sufficient for ohr induction by OHPs. Thus, the ohr gene appears to encode an antioxidant enzyme that is not part of the OxyR regulon yet is specifically induced by OHPs. (+info)
Application of rat hepatocyte culture to predict in vivo metabolic auto-induction: studies with DFP, a cyclooxygenase-2 inhibitor.
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The drug candidate DFP [5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonylphenyl)-2(5H)-furanone] is a selective cyclooxygenase-2 inhibitor under evaluation for analgesic and anti-inflammatory therapy. The in vitro metabolic pathways (rat microsomes) involve hydroxylation of the isopropyl side chain at either of two positions, the methyl or the methine, thus producing a hydroxylated metabolite (DFHP) or a dealkylated metabolite (DFH). DFH formation was the major pathway. Using hepatic microsomes from rats treated with agents that induce specific CYP isozymes, it was shown that the dexamethasone-inducible rat CYP3A isozyme(s) play a major role in DFH formation. The roles of CYP3A1 and -3A2 were confirmed with genetically engineered rat CYP enzymes. The potential for induction of rat CYP3A by DFP was evaluated by incubating DFP in rat hepatocyte cultures and measuring the CYP3A levels. Both CYP3A immunoreactive protein and enzyme activity were induced in a dose-dependent manner. The induction was confirmed in vivo by dosing rats with DFP at 100 mg/kg for 4 days. Microsomes prepared from the excised livers showed that DFP gave approximately 55% of the induction observed with dexamethasone, as determined by Western blot. In vitro metabolic auto-induction of DFP was assessed by measuring the metabolism of DFP in hepatocytes treated with DFP. DFH formation was significantly enhanced in the DFP-treated cells. In vivo, treating rats with DFP at doses of 10 to 100 mg/(kg.day) for 13 weeks indicated that DFP induced its own metabolism. The C(max) and plasma drug area under the curve values during the thirteenth week were significantly lower than that on the first day, and the effect was dose-dependent. (+info)
Identification and molecular characterization of an efflux system involved in Pseudomonas putida S12 multidrug resistance.
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The authors previously described srpABC, an operon involved in proton-dependent solvent efflux in the solvent-tolerant Pseudomonas putida S12. Recently, it was shown that organic solvents and not antibiotics induce this operon. In the present study, the authors characterize a new efflux pump, designated ArpABC, on the basis of two isolated chloramphenicol-sensitive transposon mutants. The arpABC operon is involved in the active efflux of multiple antibiotics, such as tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin and novobiocin. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins of the resistance/nodulation/cell division family, which are involved in both organic solvent and multiple drug efflux. These findings demonstrate that ArpABC is highly homologous to the MepABC and TtgABC efflux systems for organic solvents and multiple antibiotics. However, ArpABC does not contribute to organic solvent tolerance in P. putida S12 but is solely involved in multidrug resistance. (+info)
Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam.
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To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450cam, we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450cam from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm-1, indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate-binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O-O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450cam is important for the construction of the heme pocket and the heterolysis of the O-O bond. (+info)
In vitro inhibition of cytochrome P450 enzymes in human liver microsomes by a potent CYP2A6 inhibitor, trans-2-phenylcyclopropylamine (tranylcypromine), and its nonamine analog, cyclopropylbenzene.
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Currently, there are no selective, well characterized inhibitors for CYP2A6. Therefore, the effects of trans-(+/-)-2-phenylcyclopropylamine (tranylcypromine), a potent CYP2A6 inhibitor, on human liver microsomal cytochromes P450 (CYP) were studied to elucidate its selectivity. The IC50 value of tranylcypromine in coumarin 7-hydroxylation (CYP2A6 model activity) was 0.42 +/- 0.07 microM and in chlorzoxazone 6-hydroxylation (CYP2E1 model activity) 3.0 +/- 1.1 microM. The IC50 values for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activities were >10 microM. Potency and selectivity of tranylcypromine were strongly dependent on the amine group, because its nonamine analog cyclopropylbenzene was much less potent inhibitor of CYP1A, CYP2A6, CYP2C19, and CYP2E1 activities and did not inhibit at all CYP2C9, CYP2D6, or CYP3A4 activities. In human liver microsomes tranylcypromine induced type II and cyclopropylbenzene type I difference spectrum. According to the double reciprocal analysis of these spectral responses both tranylcypromine and cyclopropylbenzene may have at least two P450-related binding sites in liver microsomes. The K(a) values of tranylcypromine varied from 4.5 to 15.1 microM and -34.3 to 167 microM in microsomes derived from three different livers and of cyclopropylbenzene from -1.6 to 10.1 microM and -34.6 and 75.2 microM in the same liver microsomes. Based on these results, tranylcypromine seems an adequately selective CYP2A6 inhibitor for in vitro use. (+info)
Solute-stationary phase interaction in gas liquid chromatography. Thermodynamic parameters for substituted halogenobenzene derivatives.
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Thermodynamic parameters were determined by variable temperature experiments on the gas liquid chromatography (GLC) relative retention values, log gamma, of 3- and 4-substituted halogenobenzene derivatives under non-polar condition. These deltadeltaHdegrees(s)(R,X) had a compensational relation with the deltadeltaSdegrees(s)(R,X) values. The free energy change deltadeltaGdegrees(s)(R,X) which is estimated from the deltadeltaHdegrees(s)(R,X) and the deltadeltaSdegrees(s)(R,X) at 298 K is less than -23 kJ x mol(-1). The deltadeltaSdegrees(s)(R,X) values could be explained by translational entropy change for the adsorbed active complex between the solute and stationary phase. It is suggested that the interaction could be mainly expressed by the physical adsorption exclusive of hydrogen bonding. The deltadeltaGdegrees(s)(R,X) values for each halogen derivative have been the excellent linear lines taking the same slopes for the monosubstituted benzene derivatives, deltadeltaGdegrees(s)(R,X). The regression analyses of their intercepts were given using the descriptor evaluated as the molecular volume sigmaa(X) rather than sigmaM(X). The regression analysis for polyhalogenobenzene was also given successfully using sigmaa(Xn). (+info)
Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450(cam)).
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Polychlorinated benzenes are recalcitrant environmental pollutants primarily because they are resistant to attack by dioxygenases commonly used by micro-organisms for the biodegradation of aromatic compounds. We have investigated the oxidation of polychlorinated benzenes by mutants of the haem mono-oxygenase CYP101 (cytochrome P450(cam)) from Pseudomonas putida with the aim of generating novel systems for their biodegradation. Wild-type CYP101 had low activity for the oxidation of dichlorobenzenes and trichlorobenzenes to the chlorophenols, but no products were detected for the heavily chlorinated benzenes. Increasing the active-site hydrophobicity with the Y96F mutation increased the activity up to 100-fold, and both pentachlorobenzene and hexachlorobenzene were oxidized slowly to pentachlorophenol. Decreasing the space available at the top of the active site with the F87W mutation to force the substrate to be bound closer to the haem resulted in a further 10-fold increase in activity with most substrates. Introducing the F98W mutation, also at the top of the active site, decreased the NADH-turnover rates but increased the coupling efficiencies, and > 90% coupling was observed for 1,3-dichlorobenzene and 1,3,5-trichlorobenzene with the F87W--Y96F--F98W mutant. The V247L mutation generally increased the NADH-turnover rates, and the F87W--Y96F--V247L mutant showed reasonably fast NADH turnover (229 min(-1)) with the highly insoluble pentachlorobenzene without the need for surfactants or organic cosolvents. As all chlorophenols are degraded by micro-organisms, novel biodegradation systems could be constructed in which CYP101 mutants convert the inert polychlorinated benzenes to the phenols, which are then readily degraded by natural pathways. (+info)