Correct assembly of iron-sulfur cluster FS0 into Escherichia coli dimethyl sulfoxide reductase (DmsABC) is a prerequisite for molybdenum cofactor insertion

The FS0 [4Fe-4S] cluster of the catalytic subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase (DmsABC) plays a key role in the electron transfer relay. We have now established an additional role for the cluster in directing molybdenum cofactor assembly during enzyme maturation. EPR spectroscopy indicates that FS0 has a high spin ground state (S = 3/2) in its reduced form, resulting in an EPR spectrum with a peak at g ～ 5.0. The cluster is predicted to be in close proximity to the molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) cofactor, which provides the site of dimethyl sulfoxide reduction. Comparison with nitrate reductase A (NarGHI) indicates that a sequence of residues ((18)CTVNC(22)) plays a role in both FS0 and Mo-bisPGD coordination. A DmsA(ΔN21) mutant prevented Mo-bisPGD binding and resulted in a degenerate [3Fe-4S] cluster form of FS0 being assembled. DmsA belongs to the Type II subclass of Mo-bisPGD-containing catalytic subunits that is distinguished from the Type I subclass by having three rather than two residues between the first two Cys residues coordinating FS0 and a conserved Arg residue rather than a Lys residue following the fourth cluster coordinating Cys. We introduced a Type I Cys group into DmsA in two stages. We changed its sequence from (18)C(A)TVNC(B)GSRC(C)P(27) to (18)C(A)TYC(B)GVGC(C)G(26) (similar to that of formate dehydrogenase (FdnG)) and demonstrated that this eliminated both Mo-bisPGD binding and EPR-detectable FS0. We then combined this change with a DmsA(R61K) mutation and demonstrated that this additional change partially rescued Mo-bisPGD insertion.     

Transient kinetic studies of heme reduction in Escherichia coli nitrate reductase A (NarGHI) by menaquinol

We have studied the transient kinetics of quinol-dependent heme reduction in Escherichia coli nitrate reductase A (NarGHI) by the menaquinol analogue menadiol using the stopped-flow method. Four kinetic phases are observed in the reduction of the hemes. A transient species, likely to be associated with a semiquinone radical anion, is observed with kinetics that correlates with one of the phases. The decay of the transient species and the formation of the second reduction phase of the hemes can be fitted to a double-exponential equation giving similar rate constants, k(1) = 9.24 +/- 0.9 s(-1) and k(2) = 0.22 +/- 0.02 s(-1) for the decay of the transient species, and k(1) = 9.23 +/- 0.9 s(-1) and k(2) = 0.22 +/- 0.02 s(-1) for the formation of the reduction phase. The quinol-binding-site inhibitors 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and stigmatellin have significant and different inhibitory effects on the reduction kinetics. The kinetics of heme reduction in NarI expressed in the absence of the NarGH catalytic dimer (NarI(DeltaGH) exhibits only two kinetic phases, and the decay of the transient species also correlates kinetically with the second reduction phase of the hemes. We have also studied nitrate-dependent heme reoxidation following quinol-dependent heme reduction using a sequential stopped-flow method. HOQNO elicits a much stronger inhibitory effect than stigmatellin on the reoxidation of the hemes. On the basis of our results, we propose schemes for the mechanism of NarGHI reduction by menaquinol and reoxidation by nitrate.     

The Escherichia coli ynfEFGHI operon encodes polypeptides which are paralogues of dimethyl sulfoxide reductase (DmsABC)

The ynfEFGHI operon is a paralogue of the Escherichia coli dmsABC operon. ynfE and ynfF are paralogues of dmsA. ynfG and ynfH are paralogues of dmsB and dmsC, respectively. YnfI (dmsD) has no dms paralogue. YnfE/F and YnfG could be detected by immunoblotting with anti-DmsAB antibodies when expressed under the control of a tac or dms promoter. Cells harbouring ynfFGH on a multicopy plasmid supported anaerobic growth with dimethyl sulfoxide (DMSO) as respiratory oxidant in a dmsABC deletion, suggesting that YnfFGH forms a heterotimeric enzyme complex similar to DmsABC. Exchange of DmsC by YnfH (DmsAB-YnfH) resulted in membrane localization, anaerobic growth on DMSO, and binding of 2-n-heptyl 4-hydroxyquinoline-N-oxide, indicating that YnfH was a competent anchor. YnfG can also replace DmsB as the electron transfer subunit and assembled [Fe-S] clusters as judged by electron paramagnetic resonance spectroscopy. YnfE and/or YnfF could not form a functional complex with DmsBC and expression of YnfE prevented the accumulation of YnfFGH.     

Electron transfer from menaquinol to fumarate. Fumarate reductase anchor polypeptide mutants of Escherichia coli

Fumarate reductase (FRD) of Escherichia coli is a four-subunit membrane-bound complex that is synthesized during anaerobic growth when fumarate is available as a terminal oxidant. The two subunits that comprise the catalytic domain, FrdA and FrdB, are anchored to the cytoplasmic membrane surface by two small hydrophobic polypeptides, FrdC and FrdD, which are also required for the enzyme to interact with quinone. To better define the individual roles of the FrdC and FrdD polypeptides in FRD complex formation and quinone binding, we selectively mutagenized the frdCD genes. Frd- strains were identified by their inability to grow on restrictive media, and the resulting mutant FRD complexes were isolated and biochemically characterized. The majority of the frdC and frdD mutations were identified as single base deletions that caused premature termination in either FrdC or FrdD and resulted in the loss of one or more of the predicted transmembrane helices. Two additional frdC mutants were characterized that contained single base changes resulting in single amino acid substitutions. All mutant enzyme complexes were incapable of oxidizing the physiological electron donor, menaquinol-6, in the presence of fumarate. Additionally, the ability of the mutant complexes to oxidize reduced benzyl viologen or reduce the ubiquinone analogue 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone and phenazine methosulfate with succinate as electron donor were also affected but to varying degrees. The separation of oxidative and reductive activities with quinones suggests there are two quinone binding sites in the fumarate reductase complex and that electron transfer occurs in two le- steps carried out at these separate sites.     

Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.     

Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane.

Escherichia coli grown anaerobically on nitrate exhibited the same transport barrier to reduction of chlorate, relative to nitrate, as that exhibited by Paracoccus denitrificans. This establishes that the nitrate binding site of nitrate reductase (EC 1.7.99.4) in E. coli must also lie on the cell side of the nitrate transporter which is associated with the plasma membrane. Because nitrate reductase is membrane bound, the nitrate binding site is thus located on the inner aspect of the membrane. Nitrate pulse studies on E. coli in the absence of valinomycin showed a small transient alkalinization (leads to H+/NO3- congruent to --0.07) which did not occur with oxygen pulses. By analogy with P. denitrificans, the alkaline transient is interpreted to arise from proton-linked nitrate uptake which is closely followed by nitrite efflux. The result is consistent with internal reduction of nitrate, whereas external reduction would be expected to give leads to H+/NO3-ratios approaching --2.     

Effects of site-directed mutations on heme reduction in Escherichia coli nitrate reductase A by menaquinol: a stopped-flow study

We have studied the effects of site-directed mutations in Escherichia coli nitrate reductase A (NarGHI) on heme reduction by a menaquinol analogue (menadiol) using the stopped-flow method. For NarGHI(H66Y) and NarGHI(H187Y), both lacking heme b(L) but having heme b(H), the heme reduction by menadiol is abolished. For NarGHI(H56R) and NarGHI(H205Y), both without heme b(H) but with heme b(L), a smaller and slower heme reduction compared to that of the wild-type enzyme is observed. These results indicate that electrons from menadiol oxidation are transferred initially to heme b(L). A transient species, likely to be associated with a semiquinone radical anion, was generated not only on reduction of the wild-type enzyme as observed previously (1) but also on reduction of NarGHI(H56R) and NarGHI(H205Y). The inhibitors 2-n-heptyl-4-hydroxyquinoline-N-oxide and stigmatellin both have significant effects on the reduction kinetics of NarGHI(H56R) and NarGHI(H205Y). We have also investigated the reoxidation of menadiol-reduced heme by nitrate in the mutants. Compared to the wild type, no significant heme reoxidation is observed for NarGHI(H56R) and NarGHI(H205Y). This result indicates that a single mutation removing heme b(H) blocks the electron-transfer pathway from the subunit NarI to the catalytic dimer NarGH.     

Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli

Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and from a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of the inhibitor methotrexate (MTX). Although the Asp27----Asn substitution causes only small changes in the association rate constants (kon) for the formation of binary and ternary (with NADPH) complexes, the dissociation rate constants for these complexes (koff) are increased for the mutant enzyme by factors of about 5- and 100-fold, respectively, at pH 7.65. In binding experiments, the initial MTX binary and ternary complexes of the mutant enzyme were found to undergo relatively rapid isomerization (kobs approximately 17 and 145 s-1, respectively). Although such rapid isomerization of complexes of WT-ECDHFR could not be detected in binding experiments, evidence of a slow isomerization (k = 4 x 10(-3) s-1) of the ternary WT-ECDHFR.MTX.NADPH complex was obtained from progress of inhibition experiments. This slow isomerization increases binding of MTX to WT-ECDHFR only 2.4-fold (much less than previously estimated). From presently available data, we could not determine the contribution of the rapid isomerization of complexes to the binding of MTX to the mutant enzyme. The Asp27----Asn substitution increases the overall dissociation constant (KD) 9-fold for the binary complex and 85-fold for the ternary complex. When it is also taken into account that a proton ultimately derived from the solvent must be added to MTX bound to the WT enzyme, but not to MTX bound to the mutant enzyme, these increases in KD for the mutant enzyme correspond to decreases in binding energy for MTX of 3.9 and 5.2 kcal/mol at pH 7.65 for the binary and ternary complexes, respectively.     

Aspartate residue 142 is important for catalysis by ADP-glucose pyrophosphorylase from Escherichia coli

Structural prediction of several bacterial and plant ADP-glucose pyrophosphorylases, as well as of other sugar-nucleotide pyrophosphorylases, was used for comparison with the three-dimensional structures of two crystallized pyrophosphorylases (Brown, K., Pompeo, F., Dixon, S., Mengin-Lecreulx, D., Cambillau, C., and Bourne, Y. (1999) EMBO J. 18, 4096-4107; Blankenfeldt, W., Asuncion, M., Lam, J. S., and Naismith, J. H. (2000) EMBO J. 19, 6652-6663). This comparison led to the discovery of highly conserved residues throughout the superfamily of pyrophosphorylases despite the low overall homology. One of those residues, Asp(142) in the ADP-glucose pyrophosphorylase from Escherichia coli, was predicted to be near the substrate site. To elucidate the function that Asp(142) might play in the E. coli ADP-glucose pyrophosphorylase, aspartate was replaced by alanine, asparagine, or glutamate using site-directed mutagenesis. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of the purified mutants showed a decrease in specific activity of up to 4 orders of magnitude. Comparison of other kinetic parameters, i.e. the apparent affinities for substrates and allosteric effectors, showed no significant changes, excluding this residue from the specific role of ligand binding. Only the D142E mutant exhibited altered K(m) values but none as pronounced as the decrease in specific activity. These results show that residue Asp(142) is important in the catalysis of the ADP-glucose pyrophosphorylase from E. coli.     

DmsD is required for the biogenesis of DMSO reductase in Escherichia coli but not for the interaction of the DmsA signal peptide with the Tat apparatus

The DmsD protein is essential for the biogenesis of DMSO reductase in Escherichia coli, and binds the signal peptide of the DmsA subunit, a Tat substrate. This suggests a role as a guidance factor to target pre-DmsA to the translocase. Here, we have analysed the export of fusion proteins in which the DmsA and TorA signal peptides are fused to green fluorescent protein. Both chimeras are efficiently exported to the periplasm in wild-type E. coli cells and we show that their export efficiencies are essentially identical in a mutant lacking DmsD. An authentic Tat substrate, TMAO reductase, is also efficiently exported in the dmsD mutant. The data indicate that DmsD carries out a critical role in DMSO reductase biogenesis/assembly but is not required for the functioning of the DmsA signal peptide.     

Folding of dihydrofolate reductase from Escherichia coli

The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium-Stimulated Transport of Glutamate in Escherichia coli

Wild-type Escherichia coli B grew poorly on glutamate as the sole carbon source, except at very high concentrations of the amino acid. The addition of sodium ion markedly stimulated the growth. It had the same effect in a mutant of E. coli B selected for the ability to grow at low glutamate concentrations. Sodium ion also potentiated growth inhibition by analogues of glutamate. The uptake of glutamate by nongrowing cells of the mutant was markedly stimulated by sodium ion in the presence of an energy source, chloramphenicol, and arsenite, which retarded glutamate degradation.     

Analysis of Glutamate Exit in Escherichia coli

Escherichia coli B exhibits carrier-mediated first-order exit of glutamate with a half-time of less than 4 min, similar to that observed in K-12 strains. Glutamate exit in both B and K-12 strains is inhibited by arsenite. Practically all of the radioactivity lost during exit by K-12 cells has been accounted for as glutamate in the cell filtrate.     

E292 is important for the aminoacylation activity of Escherichia coli leucyl-tRNA synthetase

Escherichia coli leucyl-tRNA synthetase (LeuRS) has a large connecting polypeptide (CP1) inserted into its active site. It was demonstrated that the peptide bond between E292–A293 was crucial for the aminoacylation activity of E. coli LeuRS. To investigate the effect of E292 on the function of Escherichia coli LeuRS, E292 was mutated to K, F, S, D, Q and A. These mutations at 292 did not change the specific activity of the amino acid activation reaction. Though the conformational change of these mutants was not detected in CD, their aminoacylation activities were impaired to varying extents. The mutation of E to K decreased the aminoacylation activity to the largest extent. Analysis of the K_m values of these mutants for the three substrates showed that the E292 was not involved in the binding of leucine and that all mutants had stronger binding with ATP.     

Tyrosine residue 300 is important for activity and stability of branching enzyme from Escherichia coli

Branching enzyme belongs to the alpha-amylase family, which includes enzymes that catalyze hydrolysis or transglycosylation at alpha-(1,4)- or alpha-(1,6)-glucosidic linkages. In the alpha-amylase family, four highly conserved regions are proposed to make up the active site. From amino acid sequence analysis a tyrosine residue is completely conserved in the alpha-amylase family. In Escherichia coli branching enzyme, this residue (Y300) is located prior to the conserved region 1. Site-directed mutagenesis of the Y300 residue in E. coli branching enzyme was used in order to study its possible function in branching enzymes. Replacement of Y300 with Ala, Asp, Leu, Ser, and Trp resulted in mutant enzymes with less than 1% of wild-type activity. A Y300F substitution retained 25% of wild-type activity. Kinetic analysis of Y300F showed no effect on the Km value. The heat stability of Y300F was analyzed, and this was lowered significantly compared to that of the wild-type enzyme. Y300F also showed lower relative activity at elevated temperatures compared to wild-type. Thus, these results show that Tyr residue 300 in E. coli branching enzyme is important for activity and thermostability of the enzyme.     

Effects of distal point-site mutations on the binding and catalysis of dihydrofolate reductase from Escherichia coli

The importance of salt bridge interactions at the NADPH binding site of dihydrofolate reductase has been studied by using site-directed mutagenesis. The mutations R44L and H45Q respectively disrupt the ionic contacts made between the 2'-phosphate and pyrophosphoryl moiety of the coenzyme and the N-terminal region of helix C. Equilibrium fluorescence experiments indicate that while the overall binding of NADPH to both free mutants is weakened by 1.1 and 1.5 kcal/mol (H45Q and R44L, respectively), the binding of dihydrofolate and tetrahydrofolate is unaffected. Despite the similar binding energies for both mutants, the transition state for the chemical hydride step is differentially destabilized relative to wild type (0.6 and 1.8 kcal/mol for H45Q and R44L, respectively). Both stopped-flow and pre-steady-state experiments suggest that the root of this effect may lie in multiple conformations for the E-NADPH complex of R44L. The ability of both mutants to transmit their effects beyond the local environment of the NADPH pocket is manifested in several details: (1) the pKa of Asp-27 (25 A away from the sites of mutation) is elevated from 6.5 in the wild type to 7.5 and 8.4 in H45Q and R44L, respectively; (2) NADPH elevates the off rates for tetrahydrofolate from 12 s-1 in the wild type to greater than 45 s-1 in R44L; and (3) bound tetrahydrofolate decreases the affinity of the enzymes for NADPH as reflected in the Km from 2 to 40 microM for H45Q (similar to wild type) but from 8 to 5000 microM for R44L.     

Site-specific mutagenesis of dihydrofolate reductase from Escherichia coli

Two site-specific mutations of dihydrofolate reductase from Escherichia coli based on the x-ray crystallographic structure were constructed. The first mutation (His-45----Gln) is aimed at assessing the interaction between the imidazole moiety and the pyrophosphate backbone of NADPH. The second (Thr-113----Val) is part of a hydrogen bonding network that contacts the dihydrofolate substrate and may be involved in proton delivery to the N5-C6 imine undergoing reduction. The first mutation was shown to alter both the association and dissociation rate constants for the cofactor so that the dissociation constant was increased 6-40-fold. A corresponding but smaller (fourfold) effect was noted in V/K but not in V compared to the wild-type enzyme. The second was demonstrated to increase the dissociation rate constant for methotrexate 20-30-fold, and presumably dihydrofolate also, with a corresponding 20-30-fold increase in the dissociation constant. In this case an identical effect was noted on V/K but not in V relative to the native enzyme. Thus, in both mutant enzymes the decrease in binding has not been translated into a loss of catalytic efficiency.     

Iron-sulfur interconversions in the anaerobic ribonucleotide reductase from Escherichia coli

The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced [4Fe-4S]⁺ form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical. The reaction of the reduced cluster with oxygen was studied by UV-visible, EPR, NMR, and Mössbauer spectroscopies. The [4Fe-4S]⁺ form is shown to be extremely sensitive to oxygen and converted to [4Fe-4S]²⁺, [3Fe-4S]^+/0, and to the stable [2Fe-2S]²⁺ form. It is remarkable that the oxidized protein retains full activity. This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active [4Fe-4S] center. This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air. Futhermore, the [2Fe-2S] form of the protein can be converted into a [3Fe-4S] form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster. This is the first report of a 2Fe to 3Fe cluster conversion.