Chemical structures critical for the induction of FMN-dependent NADH-quinone reductase in Escherichia coli.

An FMN-dependent NADH-quinone reductase is induced in Escherichia coli by growing the cells in the presence of menadione (2-methyl-1,4-naphthoquinone). Since the properties of induced enzyme are very similar to those of NAD(P)H: (quinone-acceptor) oxidoreductase (EC 1.6.99.2), known as DT-diaphorase, from animal cells, structural requirements of quinone derivatives as an inducer of NADH-quinone reductase in E. coli were examined. Among quinone derivatives examined, it was found that 2-alkyl-1,4-quinone structure with C-3 unsubstituted or substituted with Br is critical as a common inductive signal. Michael reaction acceptors which have been reported to be strong inducers of DT-diaphorase in mouse hepatoma cells were not always effective inducers in E. coli. However, several compounds, such as 2-methylene-4-butyrolactone, methylacrylate and methyl vinyl ketone, showed a slight inductive activity. The efficient inducers of NADH-quinone reductase in E. coli contain 1,4-quinone structure as a part of the inductive signal. These compounds belong to Michael acceptors and are likely to conjugate with thiol compounds such as glutathione.     

Characterization of the cloned Escherichia coli dihydrofolate reductase

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was purified from Escherichia coli strains that carried derivatives of the multicopy recombinant plasmid, pJFM8. The results of enzyme kinetic and two-dimensional gel electrophoresis experiments showed that the cloned enzyme is indistinguishable from the chromosomal enzyme. Therefore it can be concluded that these strains are ideal for use as a source of enzyme for further studies on the biochemistry and regulation of this important enzyme. The plasmid derivatives were constructed by recloning experiments that utilized several restriction endonucleases. From the analysis both of these plasmids and the purified dihydrofolate reductase enzymes it was possible to deduce the location and orientation of the dihydrofolate reductase structural gene on the parent plasmid, pJFM8.     

Purification of NADH-ferricyanide dehydrogenase and NADH-quinone reductase from Escherichia coli membranes and their roles in the respiratory chain

The respiratory chain-linked NADH-quinone reductase (NQR) and NADH-ferricyanide dehydrogenase (NFD) were extracted from membranes of Escherichia coli by n-dodecyl octaethyleneglycol monoether detergent and purified by DEAE-Sephacel, DEAE-5PW and Bio-Gel HTP column chromatography. The purified NQR contained FAD as a cofactor, catalyzed the reduction of ubiquinone-1 (Q1) and reacted with NADH, but not with deamino-NADH (d-NADH), with an apparent Km of 48 microM. On the other hand, the purified NFD contained FMN as a cofactor, reacted with both NADH and d-NADH, and catalyzed the reduction of ferricyanide but not Q1. NFD showed a high affinity for both NADH and d-NADH with a Km of 7-9 microM. NFD was inactivated, whereas NQR was rather activated, by preincubation with an electron donor in the absence of electron acceptor. These properties were compared with those of activities observed with inverted membrane vesicles with special reference to the generation of inside-positive membrane potential (delta psi). It was found that d-NADH-reactive FMN-containing NFD is a dehydrogenase part of energy-generating NADH-quinone reductase complex. The FAD-containing NQR was very similar to that purified by Jaworowski et al. (Biochemistry (1981) 20, 2041-2047), and reduced Q1 without generating delta psi.     

Isolation and characterization of the tubular organelles induced by fumarate reductase overproduction in Escherichia coli

Strains of Escherichia coli amplifying the intrinsic membrane enzyme fumarate reductase accommodate the overproduced enzyme by increasing the amount of membrane material, in the form of intracellular tubular structures. These tubules have been observed in strains harbouring multicopy frd plasmids and in ampicillin hyper-resistant strains. A procedure has been developed for isolation of tubules nearly free of cytoplasmic membrane. Using protein A-gold labelling and optical diffraction of electron micrographs, a model for tubule structure is proposed. The tubules have a lower lipid/protein ratio than the cytoplasmic membrane, with the enzyme accounting for greater than 90% of the protein in the tubules. Both cytoplasmic membranes and tubules from amplified strains are enriched in cardiolipin and have a more fluid fatty acid composition than wild-type strains. Mutants defective in cardiolipin synthesis produce tubules in response to excess fumarate reductase, but these tubules have an altered appearance, indicating that lipid-protein interactions may be important for tubule assembly.     

Characterization of the membrane domain subunit NuoK (ND4L) of the NADH-quinone oxidoreductase from Escherichia coli

The ND4L subunit is the smallest mitochondrial DNA-encoded subunit of the proton-translocating NADH-quinone oxidoreductase (complex I). In an attempt to study the functional and structural roles of the NuoK subunit (the Escherichia coli homologue of ND4L) of the bacterial NADH-quinone oxidoreductase (NDH-1), we have performed a series of site-specific mutations on the nuoK gene of the NDH-1 operon by using the homologous recombination technique. The amino acid residues we targeted included two highly conserved glutamic acids that are presumably located in the middle of the membrane and several arginine residues that are predicted to be on the cytosolic side. All point mutants examined had fully assembled NDH-1 as detected by blue-native gel electrophoresis and immunostaining. Mutations of nearly perfectly conserved Glu-36 lead to almost null activities of coupled electron transfer with a concomitant loss of generation of electrochemical gradient. A significant diminution of the coupled activities was also observed with mutations of another highly conserved residue, Glu-72. These results may suggest that both membrane-embedded acidic residues are important for the coupling mechanism of NDH-1. Furthermore, a severe impairment of the coupled activities occurred when two vicinal arginine residues on a cytosolic loop were simultaneously mutated. Possible roles of these arginine residues and other conserved residues in the NuoK subunit for NDH-1 function were discussed.     

Characterization of two active site mutations of thioredoxin reductase from Escherichia coli

Thioredoxin reductase (TRR), a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, undergoes two sequential thiol-disulfide interchange reactions with thioredoxin during catalysis. In order to assess the catalytic role of each nascent thiol of the active site disulfide of thioredoxin reductase, the 2 cysteines (Cys-136 and Cys-139) forming this disulfide have been individually changed to serines by site-directed mutageneses of the cloned trxB gene of Escherichia coli. Spectral analyses of TRR(Ser-136,Cys-139) as a function of pH and ionic strength have revealed two pKa values associated with the epsilon 456, one of which increases from 7.0 to 8.3 as the ionic strength is increased, and a second at 4.4 which is seen only at high ionic strength. epsilon 458 of wild type TRR(Cys-136,Cys-139) and epsilon 453 of TRR(Cys-136,Ser-139) are pH-independent. A charge transfer complex (epsilon 530 = 1300 M-1 cm-1), unique to TRR(Ser-136,Cys-139), has been observed under conditions of high ammonium cation concentration (apparent Kd = 54 microM) at pH 7.6. These results suggest the assignment of Cys-139 as the FAD-interacting thiol in the reduction of thioredoxin by NADPH via thioredoxin reductase. If, as with other members of this enzyme family, the two distinct catalytic functions are each carried out by a different nascent thiol, then Cys-136 would perform the initial thiol-disulfide interchange with thioredoxin. Steady state kinetic analyses of the proteins have revealed turnover numbers of 10 and 50% of the value of the wild type enzyme for TRR(Ser-136,Cys-139) and TRR(Cys-136,Ser-139), respectively, and no changes in the apparent Km values of TR(S2) or NADPH. The finding of activity in the mutants indicates that the remaining thiol can carry out interchange with the disulfide of thioredoxin, and the resulting mixed disulfide can be reduced by NADPH via the flavin.     

Characterization of the membrane domain subunit NuoJ (ND6) of the NADH-quinone oxidoreductase from Escherichia coli by chromosomal DNA manipulation

The ND6 subunit is one of seven mitochondrial DNA-encoded subunits of the proton-translocating NADH-quinone oxidoreductase (complex I). Physiological importance of the ND6 subunit is becoming increasingly apparent because a number of mutations leading to amino acid changes in this subunit have been found to be associated with known mitochondrial diseases. Using the Escherichia coli enzyme (NDH-1), we have investigated the NuoJ subunit (the E. coli counterpart of ND6) by employing a chromosomal DNA manipulation technique. A series of point mutations was constructed directly on the nuoJ gene in the chromosome targeting at highly conserved residues. Analyses with blue-native gel electrophoresis and immunological methods revealed that, in all point mutants, the assembly of NDH-1 was normal and that the deamino-NADH-K(3)Fe(CN)(6) reductase activity of the membrane was essentially the same as that of the wild-type. However, energy-coupled NDH-1 activities were affected to varied extents. Among them, mutants of the Val-65 residue that is located in the most conserved transmembrane segment significantly lost the coupled electron-transfer activities and exhibited diminished membrane potential and proton translocation. This may suggest that Val-65 or the area around it is important for energy transduction of the coupling site 1. Together with the results on mutations related to human diseases, possible functional roles of the NuoJ subunit have been discussed.     

Characterization of intracellular DNA strand breaks induced by neocarzinostatin in Escherichia coli cells.

DNA strand breaks induced by Neocarzinostatin in Escherichia coli cells have been characterized. Radioactively labeled phage lambda DNA was introduced into lysogenic host bacteria allowing the phage DNA to circularize into superhelical molecules. After drug treatment DNA single- and double-strand breaks were measured independently after neutral sucrose gradient sedimentation. The presence of alkali-labile lesions was measured in parallel in alkaline sucrose gradients. The cell envelope provided an efficient protection towards the drug, since no strand breaks were detected unless the cells were made permeable with toluene or with hypotonic Tris buffer. In permeable cells, no double strand breaks could be detected, even at high NCS concentration (100 micrograms/ml). Induction of single-strand breaks leveled off after 15 min at 20 degrees C in the presence of 2 mM mercaptoethanol. Exposure to 0.3N NaOH doubled the number of strand breaks. No enzymatic repair of the breaks could be observed.     

Sodium-transport NADH-quinone reductase of a marineVibrio alginolyticus

The respiratory chain of a marine bacterium,Vibrio alginolyticus, required Na⁺ for maximum activity, and the site of Na⁺-dependent activation was localized on the NADH-quinone reductase segment. The Na⁺-dependent NADH-quinone reductase extruded Na⁺ as a direct result of redox reaction. It was composed of three subunits, , , and , with apparentMr of 52, 46, and 32 KDa, respectively. The reduction of ubiquinone-1 to ubiquinol proceeded via ubisemiquinone radicals. The former reaction was catalyzed by the FAD-containing subunit. This reaction showed no specific requirement for Na⁺. For the formation of ubiquinol, the presence of the subunit and the FMN-containing subunit was essential. The latter reaction specifically required Na⁺ for activity and was strongly inhibited by 2-n-heptyl-4-hydroxyquinolineN-oxide. It was assigned to the coupling site for Na⁺ transport. The mode of energy coupling of redox-driven Na⁺ pump was compared with those of decarboxylase- and ATP-driven Na⁺ pumps found in other bacteria.     

Proteins induced by anaerobiosis in Escherichia coli

The contribution of protein induction and repression to the adaptation of cells to changes in oxygen supply is only poorly understood. We assessed this contribution by measuring the levels of 170 individual polypeptides produced by Escherichia coli K-12 in cells growing aerobically or anaerobically with and without nitrate. Eighteen reached their highest levels during anaerobic growth. These 18 polypeptides include at least 4 glycolytic enzymes and pyruvate formate-lyase (beta-subunit). Most of these proteins were found at significant levels during aerobic growth and appeared to undergo metabolic regulation by stimuli other than anaerobiosis. Anaerobic induction ratios ranged from 1.8- to 11-fold, and nitrate antagonized the anaerobic induction of all of the proteins except one. The time course of synthesis of the proteins after shifts in oxygen supply revealed at least three distinct temporal patterns. These results are discussed in light of known physiological alterations associated with changes in oxygen availability.     

Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli.

Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).     

Two glutamyl-tRNA reductase activities in Escherichia coli

delta-Aminolevulinic acid (ALA) is the first committed precursor for tetrapyrrole biosynthesis. ALA formation in Escherichia coli occurs in a tRNA-dependent three-step conversion from glutamate. Glu-tRNA reductase is the key enzyme in this pathway. E. coli K12 contains two Glu-tRNA reductase activities which differ in their molecular weights. Here we describe the purification of one of these enzymes. Four different chromatographic separations yielded a nearly homogeneous protein. Its apparent molecular mass under denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation and gel filtration) is 85,000 +/- 5,000 Da. This indicates a monomeric structure for the active enzyme. Gel filtration and glycerol gradient centrifugation indicate that the other activity has a molecular mass of 45,000 +/- 5,000 Da. In the presence of NADPH both enzyme activities converted E. coli Glu-tRNA(2Glu) to glutamate 1-semialdehyde. Addition of GTP or hemin did not affect the reductase activity. Both enzymes display sequence-specific recognition of tRNA; E. coli Glu-tRNA(2Glu) is a good substrate while the Chlamydomonas reinhardtii, Bacillus subtilis, and Synechocystis Glu-tRNA(Glu) species are poorly recognized.