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1.
Results of genetic analysis of bacterial tnm mutations influencing transposition of Tn9 are presented. Five independent tnm mutations were mapped at 90,5 min of the E. coli genetic map. The tnm mutations were 3,5 and 46,5% contransducible with metA and malB markers, respectively. Two tnm mutations tested were recessive in tnm+/tnm- merodiploids. The effect of tnm mutations on other transposons--Tn10, Tn601, Tn3 and Tn5 was examined. It was shown that tnm1 and tnm2 mutations reduced the frequency of transposition of Tn10, Tn3, Tn5 and Tn601 from the genome of phage lambda and inhibited intracellular development of the infecting Mu phage. The latter effect was probably due to the inhibition of Mu integration into bacterial chromosome. The tnm3 mutation affected the transposition of Tn9 only. 相似文献
2.
Recent time-resolved optical absorption studies in our laboratory have indicated that the putative peroxy intermediate formed during the reduction of dioxygen to water by cytochrome oxidase (P(R)) is a pH-dependent mixture of compound A, P, and F [Van Eps, N., et al. (2003) Biochemistry 42, 5065-5073]. This conclusion is based on a kinetic analysis of flow-flash time-resolved data using a unidirectional sequential scheme with five apparent lifetimes. To account for this observation, we propose a more complex kinetic model that consists of branched pathways, one branch producing the 607 nm P form and the other the 580 nm F form. The two pathways are interconnected, and the rate of exchange between the two is pH-dependent. The kinetic analysis and testing of the new model involves a novel algebraic approach which transforms the intermediates of the complex branched scheme into intermediates comparable to those derived on the basis of a sequential model. The branched model reproduces the experimental data very well and is consistent with a variety of experimental observations. The two branches may arise from two structurally different CO or O(2) conformers or protein conformers, which could lead to different accessibilities of proton donors to the binuclear center. 相似文献
3.
Lipid binding by Escherichia coli pyruvate oxidase is disrupted by small alterations of the carboxyl-terminal region 总被引:2,自引:0,他引:2
Escherichia coli pyruvate oxidase is a membrane-associated flavoprotein dehydrogenase which is greatly activated by lipids and detergents. The carboxyl-terminal region of the protein has been shown to play a critical role in the interaction with lipids. We report mutations generated by chemical and oligonucleotide-mediated site-directed mutagenesis of the poxB gene which result in enzymes defective in lipid activation. Nine mutants were isolated which encode enzymes with point mutations in the carboxyl-terminal segment of the protein. Two mutant lesions introduced termination codons giving enzymes lacking the last nine or three amino acids of the protein which were unable to interact with detergents in vitro and were unable to function in vivo. Of the missense mutants isolated, two were most informative. One was the substitution of Glu-564 with proline in the PoxB16 oxidase. This residue lies in the center of a putative lipid-binding amphipathic alpha-helix (Arg-558 to Thr-568) located close to the carboxyl terminus. Strains producing the PoxB16 oxidase were devoid of oxidase activity in vivo, the enzymes could not be activated by Triton X-100, and were activated poorly by phospholipids in vitro. These results indicated that the PoxB16 oxidase lacked normal lipid-binding ability. Another mutant oxidase (PoxB15) in which proline was substituted for Asp-560 at the beginning of the amphipathic alpha-helix had normal oxidase activity. These findings indicate that the amphipathic alpha-helix structure plays an essential role in the activation and lipid-binding properties of the enzyme. The second informative missense mutation was the substitution of the carboxyl-terminal arginine with glycine. This enzyme showed normal activation in vitro by phospholipids and some detergents, and somewhat reduced activity in vivo. This mutant enzyme appeared to dissociate from detergent vesicles more readily than does the normal enzyme. A model for the membrane interaction of the carboxyl terminus based on the properties of these mutant proteins is presented. 相似文献
4.
Outten FW Outten CE Hale J O'Halloran TV 《The Journal of biological chemistry》2000,275(40):31024-31029
Because copper ions are both essential cofactors and cytotoxic agents, the net accumulation of this element in a cell must be carefully balanced. Depending upon the cellular copper status, copper ions must either be imported or ejected. CopA, the principal copper efflux ATPase in Escherichia coli, is induced by elevated copper in the medium, but the copper-sensing regulatory factor is unknown. Inspection of the copA promoter reveals signature elements of promoters controlled by metalloregulatory proteins in the MerR family. These same elements are also present upstream of yacK, which encodes a putative multi-copper oxidase. Homologues of YacK are found in copper resistance determinants that facilitate copper efflux. Here we show by targeted gene deletion and promoter fusion assays that both copA and yacK are regulated in a copper-responsive manner by the MerR homologue, ybbI. We have designated ybbI as cueR for the Cu efflux regulator. This represents the first example of a copper-responsive regulon on the E. coli chromosome and further extends the roles of MerR family members in prokaryotic stress response. 相似文献
5.
R. Metheringham K. L. Tyson H. Crooke D. Missiakas S. Raina J. A. Cole 《Molecular genetics and genomics : MGG》1996,253(1-2):95-102
The assembly of anaerobically induced electron transfer chains in Escherichia coli strains defective in periplasmic disulphide bond formation was investigated. Strains deficient in DsbA, DsbB or DipZ (DsbD) were unable to catalyse formate-dependent nitrite reduction (Nrf activity) or synthesize any of the known c-type cytochromes. The Nrf+ activity and cytochrome c content of mutants defective in DsbC, DsbE or DsbF were similar to those of the parental, wild-type strain. Neither DsbC expressed from a multicopy plasmid nor a second mutation in dipZ (dsbD) was able to compensate for a dsbA mutation by restoring nitrite reductase activity and cytochrome c synthesis. In contrast, only the dsbB and dipZ (dsbD) strains were defective in periplasmic nitrate reductase activity, suggesting that DsbB might fulfil an additional role in anaerobic electron transport. Mutants defective in dipZ (dsbD) were only slightly more sensitive to Cu++ ions at concentrations above 5?mM than the parental strain, but strains defective in DsbA, DsbB, DsbC, DsbE or DsbF were unaffected. These results are consistent with our earlier proposals that DsbA, DsbB and DipZ (DsbD) are part of the same pathway for ensuring that haem groups are attached to the correct pairs of cysteine residues of apocytochromes c in the E. coli periplasm. However, neither DsbE nor DsbF are essential for the reduction of DipZ (DsbD). 相似文献
6.
Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by approximately 3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 A X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state. 相似文献
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Fourteen Tn5-generated mutations of the Rac prophage, called sbc because they suppress recB21 recC22, were found to fall into two distinct types: type I mutations, which were insertions of Tn5, and type II mutations, which were insertions of IS50. Both orientations of Tn5 and IS50 were represented among the mutants and were arbitrarily labeled A and B. All 14 of the Tn5 and IS50 insertions occurred in the same location (+/- 100 base pairs) approximately 5.6 kilobases from one of the hybrid attachment sites. Eleven of the mutants contained essentially the same amount of exonuclease VIII, the product of recE. The possibility that a promoter for recE was created by the insertion of Tn5 and IS50 was considered. Two IS50 mutants in which such a promoter could not have been created showed three to four times as much exonuclease VIII, and another showed one-half as much as the majority. The possibility was considered that a promoter internal to IS50 is responsible for this heterogeneity. Restriction alleviation was measured in all 14 mutants. An insertion of the transposon Tn10 which reduces expression of exonuclease VIII (recE101::Tn10) was located within the Rac prophage at a position 2.35 kilobases from the left hybrid attachment site. Location and orientation of the Rac prophage on the Escherichia coli genetic map are discussed in light of these results. 相似文献
10.
Excited state intramolecular proton transfer (ESIPT) reaction along the O-H[Symbol: see text][Symbol: see text][Symbol: see text][Symbol: see text]O hydrogen bond of o-hydroxy benzaldehyde (OHBA), methyl salicylate (MS) and salicylic acid (SA) was investigated by ab-initio quantum chemical calculation and theory of atoms and molecules (QTAIM) for the first time. Variation in several geometric as well as QTAIM parameters along the reaction coordinate was monitored in the fully relaxed excited state potential energy curve (PEC) obtained from intrinsic reaction coordinate (IRC) analysis. Although, the excited state barrier height for the forward reaction (?E (0) (#) ) reduces substantially in all the systems, MS and SA do not show any obvious asymmetry for proton transfer. For MS and SA, the crossover of the bond index as well as the lengths of the participating bonds at the saddle point is assigned due to this symmetry in accordance with bond energy - bond order (BEBO) model, which does not hold true in OHBA both in the ground and excited states. Bond ellipticity, covalent and metallic character were examined for different structures along the reaction path within the QTAIM framework. The QTAIM analysis was found to be able to uniquely distinguish between the ground and excited states of the OHBA molecule as well as both determining the effects on the bonding character of adding different substituent groups and differentiating between the ESIPT reactions in the SA and MS molecules. 相似文献
11.
The active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two redox active cysteine residues in the sequence -C11-P-Y-C14-H-. The 1H NMR resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cys-11 thiolate, which is shown to have an abnormally low pKa value. A hydrogen bond would also agree with activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys-11 thiolate. 相似文献
12.
Parrini C Bemporad F Baroncelli A Gianni S Travaglini-Allocatelli C Kohn JE Ramazzotti M Chiti F Taddei N 《Journal of molecular biology》2008,379(5):1107-1118
The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (kFH2O = 72,600 s− 1), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms. 相似文献
13.
Cytochrome bo is the heme-copper terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli, and functions as a redox-coupled proton pump. As an extension to our mutagenesis and Fourier-transform infrared studies on ion pumps, we examined the effects of subunit I mutations on redox-linked protein structural changes in cytochrome bo. Upon photo-reduction in the presence of riboflavin, Y288F and H333A showed profound effects in their peptide backbone vibrations (amide-I and amide-II), probably due to the loss of CuB or replacement of high-spin heme o with heme B. In the frequency region of protonated carboxylic C=O stretching vibrations, negative 1,743 cm-1 and positive 1,720 cm-1 bands were observed in the wild-type; the former shifted to 1,741 cm-1 in E286D but not in other mutants including D135N. This suggests that Glu286 in the D-channel is protonated in the air-oxidized state and undergoes hydrogen bonding changes upon reduction of the redox metal centers. Two pairs of band shifts at 2,566 (+)/2,574 (-) and 2,546 (+)/2,556 (-) cm-1 in all mutants indicate that two cysteine residues not in the vicinity of the metal centers undergo redox-linked hydrogen bonding changes. Cyanide had no effect on the protein structural changes because of the rigid local protein structure around the binuclear center or the presence of a ligand(s) at the binuclear center, and was released from the binuclear center upon reduction. This study establishes that cytochrome bo undergoes unique redox-linked protein structural changes. Localization and time-resolved analysis of the structural changes during dioxygen reduction will facilitate understanding of the molecular mechanism of redox-coupled proton pumping at the atomic level. 相似文献
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15.
Uracil DNA glycosylase (UDG) cleaves the glycosidic bond of deoxyuridine in DNA using a hydrolytic mechanism, with an overall catalytic rate enhancement of 10(12)-fold over the solution reaction. The nature of the enzyme-substrate interactions that lead to this large rate enhancement are key to understanding enzymatic DNA repair. Using (1)H and heteronuclear NMR spectroscopy, we have characterized one such interaction in the ternary product complex of Escherichia coli UDG, the short (2.7 A) H bond between His187 N(epsilon)(2) and uracil O2. The H bond proton is highly deshielded at 15.6 ppm, indicating a short N-O distance and exhibits a solvent exchange rate that is 400- and 10(5)-fold slower than free imidazole at pH 7.5 and pH 10, respectively. Heteronuclear NMR experiments at neutral pH show that this H bond involves the neutral imidazole form of His187 and the N1-O2 imidate form of uracil. The excellent correspondence of the pK(a) for the disappearance of the H bond (pK(a) = 6.3 +/- 0.1) with the previously determined pK(a) = 6.4 for the N1 proton of enzyme-bound uracil indicates that the H bond requires negative charge on uracil O2 [Drohat, A. C., and Stivers, J. T. (2000) J. Am. Chem. Soc. 122, 1840-1841]. Although the above characteristics suggest a short strong H bond, the D/H fractionation factor of phi = 1.0 is more typical of a normal H bond. This unexpected observation may reflect a large donor-acceptor pK(a) mismatch or the net result of two opposing effects on vibrational frequencies: decreased N-H bond stretching frequencies (phi < 1) and increased bending frequencies (phi > 1) relative to the O-H bonds of water. The role of this H bond in catalysis by UDG and several approaches to quantify the H bond energy are discussed. 相似文献
16.
Threonine-978 in the transmembrane segment of the multidrug efflux pump AcrB of Escherichia coli is crucial for drug transport as a probable component of the proton relay network
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Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals and expels them from cells. It is a proton antiporter belonging to the resistance-nodulation-division (RND) superfamily. Asp407, Asp408, Lys940, and Arg971 in transmembrane (TM) helices of this transporter have been identified as essential amino acid residues that probably function as components of the proton relay system. In this study, we identified a novel residue in TM helix 11, Thr978, as an essential residue by alanine scanning mutagenesis. Its location close to Asp407 suggests that it is also a component of the proton translocation pathway, a prediction confirmed by the similar conformations adopted by T978A, D407A, D408A, and K940A mutant proteins (see the accompanying paper). Sequence alignment of 566 RND transporters showed that this threonine residue is conserved in about 96% of cases. Our results suggest the hypotheses that Thr978 functions through hydrogen bonding with Asp407 and that protonation of the latter alters the salt bridging and hydrogen bonding pattern in the proton relay network, thus initiating a series of conformational changes that ultimately result in drug extrusion. 相似文献
17.
Hyperproduction of tryptophan by Escherichia coli: genetic manipulation of the pathways leading to tryptophan formation. 总被引:3,自引:1,他引:2
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Conversion of glucose and ammonium salts into tryptophan by mutants of Escherichia coli was examined as part of a feasibility study on the manufacture of tryptophan. This involved construction, largely by transduction, or a variety of multiple-mutation strains with defined genotypes. By comparing the properties of these strains, we were able to define in biochemical terms several changes that significantly enhance process productivity, namely (i) release of the first enzyme of the common pathway of aromatic biosynthesis and the first enzyme of the tryptophan pathway (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and the anthranilate aggregate, respectively) from inhibition by end products, (ii) blockage of the diversion of chorismate to phenylalanine and tyrosine biosynthesis, and (iii) presence of highly elevated tryptophan pathway enzyme levels, such as result from interference with both repression and attenuation, combined with gene amplification. By using strains carrying appropriate mutations to effect all of these changes, high values of specific productivity were obtained in bath culture (approximately 80 mg/g [dry weight] per h). Furthermore, a pronounced decay in the level of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase activity was implicated as a cause of declining process producitivity during stationary phase, emphasizing the value of having derepressed levels of this enzyme. 相似文献
18.
Conversion of glucose and ammonium salts into tryptophan by mutants of Escherichia coli was examined as part of a feasibility study on the manufacture of tryptophan. This involved construction, largely by transduction, or a variety of multiple-mutation strains with defined genotypes. By comparing the properties of these strains, we were able to define in biochemical terms several changes that significantly enhance process productivity, namely (i) release of the first enzyme of the common pathway of aromatic biosynthesis and the first enzyme of the tryptophan pathway (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and the anthranilate aggregate, respectively) from inhibition by end products, (ii) blockage of the diversion of chorismate to phenylalanine and tyrosine biosynthesis, and (iii) presence of highly elevated tryptophan pathway enzyme levels, such as result from interference with both repression and attenuation, combined with gene amplification. By using strains carrying appropriate mutations to effect all of these changes, high values of specific productivity were obtained in bath culture (approximately 80 mg/g [dry weight] per h). Furthermore, a pronounced decay in the level of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase activity was implicated as a cause of declining process producitivity during stationary phase, emphasizing the value of having derepressed levels of this enzyme. 相似文献
19.
The mechanism of enzymatic elongation by Escherichia coli DNA polymerase II of a DNA primer, which is annealed to a unique position on the bacteriophage fd viral DNA, has been studied. The enzyme is found to dissociate from the substrate at specific positions on the genome which act as “barriers” to further primer extension. It is believed these are sites of secondary structure in the DNA. When the template is complexed with E. coli DNA binding protein many of these barriers are eliminated and the enzyme remains associated with the same primer-template molecule during extensive intervals of DNA synthesis. Despite the presence of E. coli DNA binding protein, at least one barrier on the fd genome remains rate-limiting to chain extension and disturbs the otherwise processive mechanism of DNA synthesis. This barrier is overcome by increasing the concentration of enzyme.In contrast, it is found that DNA polymerase I is not rate-limited by structural barriers in the template, however, it exhibits a non-processive mechanism of elongation.These findings provide a framework for understanding the necessity for participation of proteins other than a DNA polymerase in chain extension during chromosomal replication. 相似文献
20.
Isolated Escherichia coli ribosomal protein L11 was labeled with maleimidyl derivatives of coumarin or fluorescein at the thiol group of its single cysteine, then reconstituted singly or in pairs with other fluorescently labeled ribosomal components. The characteristics of fluorescence from the labeled protein were studied and its distance to other components was determined by non-radiative energy transfer. The distance between probes on L11 and cysteine residues on other proteins or the 3' end of the ribosomal RNAs were found to be: S1, 7.4-8.3 nm; S21, 7.6 nm; 23S RNA, 6.9 nm; 5S RNA, 7.6 nm; 16S RNA, greater than 8.5 nm. Considered together with previously published results these distances indicate that the location of L11 in the 50S subunit is below the lateral protuberance characterized by L7/L12. 相似文献