Characterization by 1H NMR of a C32S,C35S double mutant of Escherichia coli thioredoxin confirms its resemblance to the reduced wild-type protein

A mutant of Escherichia coli thioredoxin containing serine residues in place of the two active-site cysteines, termed C32S,C35S, previously shown to be partially able to substitute for reduced thioredoxin in certain phage systems, has been characterized by 1H NMR spectroscopy at pH values between 5.5 and 10. The 1H NMR spectrum of the mutant at pH 5.5 is very similar to that of the wild-type protein in either the reduced or oxidized state. Chemical shift changes in the vicinity of the active site serines indicate that the nearby hydrophobic pocket is somewhat changed, probably as a result of the replacement of the cysteine thiols with the smaller, more hydrophilic hydroxyl side chains and a change in the preferred chi 1 angles of the side chains. Although the pattern of amide protons persistent in 2H2O differs only slightly between the two forms of the wild-type protein, the pattern observed for the C32S,C35S mutant shows characteristic features that correspond closely with those of the reduced wild-type protein rather than with the oxidized form. The pH dependence of the mutant protein shows a single group titrating close to the active site with a pKa of 8.3, which we assign to the buried carboxyl group of Asp 26 by analogy with the behavior of wild-type thioredoxin. The pKa is significantly higher for the mutant protein, consistent with an increase in the hydrophobicity of the pocket where the carboxyl is buried, probably due to repacking caused by the removal of the cysteine thiols and the placement of the serine hydroxyls in positions where they interact better with solvent. The results demonstrate that the solution behavior of the mutant protein is similar in many ways to that of reduced wild-type thioredoxin, explaining its partial activity in the two essential roles of reduced thioredoxin as a subunit of phage T7 DNA polymerase and in the assembly of filamentous phage.     

NMR studies of the Escherichia coli Trp repressor.trpRs operator complex

To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 1H-NMR spectroscopy. We report here the 1H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor. Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor. Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence. The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules. We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex. The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA. This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator.     

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli

The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3-->Glu, Lys9--> Glu, Arg26-->Asp and Arg124-->Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity. The acidic pI (approximately 5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.     

Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin

Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.     

Isolation and certain properties of mutant alkaline phosphatase of Escherichia coli

Natural and mutant alkaline phosphatases with amino acid substitutions in the processing site and N-terminal domain of the mature polypeptide chain Val for Ala(-1), Gln for Glu (+4) and simultaneously Gln for Glu (+4) and Ala for Arg (+1) have been isolated from the periplasm and cultural fluid of E. coli. It has been found that these substitutions have little effect on the dependence of the enzyme activity on pH, ionic strength and temperature but influence its isoenzymic spectrum and decrease (almost twofold) the maximal rate of the enzyme-catalyzed reaction. Extracellular enzymes display, in contrast with periplasmic ones, other catalytic properties (Vmax) and binding activity (Km). After translocation through the outer membrane all the enzymes display decreased Vmax and increased Km. These changes are especially well-pronounced in case of the mutant protein PhoA46 which contains an uncleaved signal peptide due to the impossibility of processing resulting from the substitution of Val for Ala(-1). The Vmax for this protein is decreased 20 times, while the Km is increased 4-fold. The protein also shows a higher (in comparison with other proteins) sensitivity towards proteolytic enzymes and is less resistant upon storage. The experimental data suggest that the changes in the N-end of alkaline phosphatase located at a long distance from its active center influence the enzyme function.     

General acid/base catalysis in the active site of Escherichia coli thioredoxin

Enzymic catalysts of thiol:disulfide oxidoreduction contain two cysteine residues in their active sites. Another common residue is an aspartate (or glutamate), the role of which has been unclear. Escherichia coli thioredoxin (Trx) is the best characterized thiol:disulfide oxidoreductase, and in Trx these three active-site residues are Cys32, Cys35, and Asp26. Structural analyses had indicated that the carboxylate of Asp26 is positioned properly for the deprotonation of the thiol of Cys35, which would facilitate its attack on Cys32 in enzyme-substrate mixed disulfides. Here, Asp26 of Trx was replaced with isologous asparagine and leucine residues. D26N Trx and D26L Trx are reduced and oxidized more slowly than is wild-type Trx during catalysis by E.coli thioredoxin reductase. Stopped-flow spectroscopy demonstrated that the cleavage of the mixed disulfide between Trx and a substrate is slower in the D26N and D26L enzymes. Buffers increase the rate of mixed disulfide cleavage in these variants but not in wild-type Trx. These results indicate that Asp26 serves as an acid/base in the oxidation/reduction reactions catalyzed by Trx. Specifically, Asp26 protonates (during substrate oxidation) or deprotonates (during substrate reduction) the thiol of Cys35. A similar role is likely filled by the analogous aspartate (or glutamate) residue in protein disulfide isomerase, DsbA, and other thiol:disulfide oxidoreductases. Moreover, these results provide the first evidence for general acid/base catalysis in a thiol:disulfide interchange reaction.     

NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: implications for the enzymatic mechanism

Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein disulfide groups and glutathione-containing mixed disulfide groups via an active site Grx-glutathione mixed disulfide (Grx-SG) intermediate. The NMR solution structure of the Escherichia coli Grx3 mixed disulfide with glutathione (Grx3-SG) was determined using a C14S mutant which traps this intermediate in the redox reaction. The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular beta-bridge between the protein and glutathione. The solution structure of E. coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specificity of reduction observed in Grxs. Molecular details of the structure in relation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed. A comparison of glutathione binding in Grx3-SG and ligand binding in other members of the thioredoxin superfamily is presented, which illustrates the highly conserved intermolecular interactions in this protein family.     

Isolation and in vitro characterization of CheZ suppressors for the Escherichia coli chemotactic response regulator mutant CheYN23D

The phosphorylated form of the response regulator CheY promotes the tumble signal in Escherichia coli chemotaxis. Phospho-CheY is thought to interact with the switch at the base of the flagellar motor and cause reversal of flagellar rotation from counterclockwise to clockwise changing the swimming direction. Thus the level of phospho-CheY controls the direction of flagellar rotation. The decay of the tumble signal is caused by dephosphorylation of CheY. CheY has an intrinsic autophosphatase activity; however, this reaction is greatly accelerated by the presence of the CheZ protein. We have shown previously that mutations at residues Asn-23 and Lys-26 in CheY confer resistance to the dephosphorylation activity of CheZ (Sann, M.G., Swanson, R.V., Bourret, R.B., and Simon, M.I. (1995) Mol. Microbiol. 15, 1069-79). Here we show that mutant CheY(N23D) is impaired in binding to CheZ, which provides a possible explanation for its resistance to the dephosphorylation activity of CheZ. Moreover, we isolated CheZ second-site suppressors of CheY(N23D), which restore both dephosphorylation and binding activity in a CheY(N23D) background. When the CheZ suppressor mutations are mapped, they are found in two clusters at the N and C termini of the CheZ protein which could define two regions of interaction with CheY. Furthermore, these regions may generate a surface in the folded three-dimensional structure of CheZ required for interaction with CheY.     

Structural determinants of the catalytic reactivity of the buried cysteine of Escherichia coli thioredoxin

The structurally homologous thioredoxins and thioltransferases/glutaredoxins possess a solvent-exposed cysteine sulfur which carries out a nucleophilic attack on the target disulfide as well as a structurally adjacent solvent inaccessible thiol. The mechanistic basis of the essentially exclusive redox reactivity of the thioredoxins in contrast to the thiol-disulfide exchange reactions characteristic of the thioltransferases lies in the relative reactivity of the buried cysteine. A stable analog of the mixed disulfide state of Escherichia coli thioredoxin is used to demonstrate a pK value of 11.1 for the solvent inaccessible Cys 35 thiol. NMR chemical shift pH titration analysis indicates a very low dielectric surrounding the Cys 35 sulfur providing a basis for both the elevated pK and the enhanced apparent nucleophilicity. The buried Asp 26 likely serves as the proton sink for the (de)protonation of Cys 35. Relevance to the reactivity of the mammalian protein isomerases is discussed.     

Expression of mutant horse cytochrome c genes in Escherichia coli

Here we describe genetically engineered constructs for the expression in Escherichia coli of genes for horse cytochrome c mutants. These constructs allow the expression of the cytochrome c genes together with hemeligase, an enzyme which covalently links heme to cytochrome. Careful selection of producer strains and the adjustment of the conditions of expression provided for expression levels of 10-15 mg of protein per liter of culture. This is by an order of magnitude greater than the expression previously achieved in yeast. A series of horse cytochrome c mutants were obtained in this way.     

A 30 S precursor of 30 S ribosomes in a mutant of Escherichia coli

Escherichia coli 15-28, a mutant with a defect in ribosome metabolism, accumulates a ribonucleoprotein particle that is indistinguishable from 30S subunits by sedimentation but contains the precursor form of 16S RNA. This particle is probably a precursor of 30 S ribosomes.     

Colonization ability and pathogenic properties of a fim- mutant of an avian strain of Escherichia coli

Several studies suggest that the expression of type 1 fimbriae is involved in the virulence of Escherichia coli in chickens, by promoting adhesion of bacteria to the respiratory tract, which is most probably the first step to occur in the infection, and by interacting with the immune response. In order to determine to what extent type 1 fimbriae were involved in the pathogenic process, the fim cluster of an avian pathogenic strain of E. coli, MT78 (O2:K1:H+), was modified in vitro and reintroduced in the parent strain via allele exchange using suicide vector pCVD442. The mutant strain thus generated (DM34) had its entire fim cluster removed. Its pathogenic properties were compared to those of the parent strain in an experimental reproduction of avain colibacillosis in 15-day-old chickens, after primary infection with infectious bronchitis virus followed by intratracheal inoculation of the challenge strain. In specific-pathogen-free (SPF) animals, mutant DM34 was less pathogenic than the parent strain and colonized the lungs of infected animals to a lower level. In germ-free chickens, although DM34 was less pathogenic than MT78 according to the differences in weight gains, it colonized the trachea, lungs and internal organs to the same extent as MT78. Our results suggest that, whereas type 1 fimbriae are not strictly required in colonization of the respiratory tract of germ-free chickens, they might be important in establishing a bacterial population in the lungs of SPF animals. The difference regularly observed in weight gains between mutant- and wild-type-inoculated chickens reflects a decreased pathogenicity of the fim- mutant. However, the isolation of E. coli in target organs and the observation of colibacillosis symptoms and lesions in mutant-inoculated chickens suggest that type 1 fimbriae by themselves play a limited role in pathogenicity.     

Role of electrostatic interactions on the affinity of thioredoxin for target proteins. Recognition of chloroplast fructose-1, 6-bisphosphatase by mutant Escherichia coli thioredoxins

Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1, 6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme. Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin. To examine the contribution of long range electrostatic interactions to the thiol/disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E. coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines. After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants. However, the conversion of cysteine residues back to cystine depended on the target protein. Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts. Moreover, the affinity of E30K, L94K, and E30K/L94K E. coli thioredoxin for chloroplast fructose-1,6-bisphosphatase (A0.5 = 9, 7, and 3 microM, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A0.5 = 33 microM), though still lower than that of thioredoxin-f (A0.5 = 0.9 microM). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A0.5 for all bacterial variants to a limiting value of approximately 9 microM but also increases the A0.5 of chloroplast thioredoxin-f. While negatively charged chloroplast fructose-1,6-bisphosphatase (pI = 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI = 11.2) diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange.     

Use of a DNA polymerase III bypass mutant of Escherichia coli, pcbA1, to isolate potentially useful mutations of a complex plasmid replicon

We report a case of a 63-year-old woman who presented with pseudoaneurysm of the free wall of the left ventricle secondary to myocardial infarction, in the presence of angiographically normal major coronary arteries. This is the only such case we know of, in which the patient underwent successful surgical correction. At last follow-up, the patient was in good condition with no evidence of cardiac disease, at 9 years after surgery.     

Characterization of Escherichia coli thioredoxin variants mimicking the active-sites of other thiol/disulfide oxidoreductases

Thiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys-Xaa-Xaa-Cys (Xaa corresponds to any amino acid). Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000-fold difference in the equilibrium constant (K(eq)) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin. Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa-Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild-type, with the order wild-type < PDI-type < DsbA-type < glutaredoxin-type. The most oxidizing, glutaredoxin-like variant has a 420-fold decreased value of K(eq), corresponding to an increase in redox potential by 75 mV. While oxidized wild-type thioredoxin is more stable than the reduced form (delta deltaG(ox/red) = 16.9 kJ/mol), both redox forms have almost the same stability in the variants. The pH-dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active-site thiol (Cys32). A pKa of 7.1 was measured for Cys32 in the reduced wild-type. All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin-like variant. A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level. However, the predicted correlation between the measured delta deltaG(ox/red) values and Cys32 pKa values was only qualitative.     

NMR assignments, secondary structure and hydration of oxidized Escherichia coli flavodoxin

Recombinant flavodoxin from Escherichia coli was uniformly enriched with 15N and 13C isotopes and its oxidized form in aqueous solution investigated by three-dimensional NMR spectroscopy. Nearly complete 1H, 15N and 13C resonance assignments were obtained. The secondary structure was determined from chemical shift, NOE and 3J(HNH alpha) coupling constant data. Like homologous long-chain flavodoxins, E. coli flavodoxin contains a five-stranded parallel beta-sheet and five helices. The beta-strands were found to comprise the residues 3-8, 29-34, 48-56, 80-89, 114-116 and 141-145. The helices comprise residues 12-25, 40-45, 62-73, 98-108 and 152-166. The FMN-binding site was determined by intermolecular NOEs and low-field shifted amide proton resonances induced by the phosphoester group of the cofactor. The data are in good agreement with a previously predicted model of E. coli flavodoxin [Havel, T. F. (1993) Mol. Sim. 10, 175-210]. The analysis of of water-flavodoxin NOEs revealed the presence of two, possibly three, buried hydration water molecules which are located at sites, where homologous flavodoxins from Anacystis nidulans and Anabena 7120 contain conserved hydration water molecules. One of these water molecules mediates hydrogen bonds between the protein backbone and the ribityl chain of the FMN cofactor.     

Purification and characterization of an isoaspartyl dipeptidase from Escherichia coli

We have identified a gene (iadA) in Escherichia coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-beta-aspartyl, dipeptides. We demonstrate at least a 3000-fold purification of the enzyme to homogeneity from crude cytosol. From the amino-terminal amino acid sequence obtained from this preparation, we designed an oligonucleotide that allowed us to map the gene to the 98-min region of the chromosome and to clone and obtain the DNA sequence of the gene. Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases, while a marked similarity was found with several dihydroorotases and imidases, reflecting the similarity in the structures of the substrates for these enzymes. Using an E. coli strain containing a plasmid overexpressing this gene, we were able to purify sufficient amounts of the dipeptidase to characterize its substrate specificity. We also examined the phenotype of two E. coli strains where this isoaspartyl dipeptidase gene was deleted. We inserted a chloramphenicol cassette into the disrupted coding region of iadA in both a parent strain (MC1000) and a derivative strain (CL1010) lacking pcm, the gene encoding the L-isoaspartyl methyltransferase involved in the repair of isomerized proteins. We found that the iadA deletion does not result in reduced stationary phase or heat shock survival. Analysis of isoaspartyl dipeptidase activity in the deletion strain revealed a second activity of lower native molecular weight that accounts for approximately 31% of the total activity in the parent strain MC1000. The presence of this second activity may account for the absence of an observable phenotype in the iadA mutant cells.