Mapping the surface of Escherichia coli peptide deformylase by NMR with organic solvents

Identifying potential ligand binding sites on a protein surface is an important first step for targeted structure-based drug discovery. While performing control experiments with Escherichia coli peptide deformylase (PDF), we noted that the organic solvents used to solubilize some ligands perturbed many of the same resonances in PDF as the small molecule inhibitors. To further explore this observation, we recorded (15)N HSQC spectra of E. coli peptide deformylase (PDF) in the presence of trace quantities of several simple organic solvents (acetone, DMSO, ethanol, isopropanol) and identified their sites of interaction from local perturbation of amide chemical shifts. Analysis of the protein surface structure revealed that the ligand-induced shift perturbations map to the active site and one additional surface pocket. The correlation between sites of solvent and inhibitor binding highlights the utility of organic solvents to rapidly and effectively validate and characterize binding sites on proteins prior to designing a drug discovery screen. Further, the solvent-induced perturbations have implications for the use of organic solvents to dissolve candidate ligands in NMR-based screens.     

Evidence that peptide deformylase and methionyl-tRNA(fMet) formyltransferase are encoded within the same operon in Escherichia coli.

Overexpression of the fms gene, the first translation unit of a dicistronic operon that also encodes methionyl-tRNA(fMet) formyltransferase in Escherichia coli, sustains the overproduction of peptide deformylase activity in crude extracts. This suggests that the fms gene encodes the peptide deformylase. Moreover, the fms gene product has a motif characteristic of metalloproteases, an activity compatible with deformylase. The corresponding protein could be purified to homogeneity. However, its enzymatic activity could not be retained during the purification procedure. As could be expected from the occurrence in its amino acid sequence of a zinc-binding motif characteristic of metallopeptidases, the purified fms product displayed one tightly bound zinc atom.     

Escherichia coli SlyD, more than a Ni(II) reservoir

SlyD interacts with HypB and contributes to nickel insertion during [NiFe]-hydrogenase biogenesis. Herein, we provide evidence of SlyD acting as a nickel storage determinant in Escherichia coli and show that this Ni(II) can be mobilized to HypB in vitro even under competitive conditions. Furthermore, SlyD enhances the GTPase activity of HypB, and acceleration of release of Ni(II) from HypB is more pronounced when HypB is GDP-bound. The data support a model in which a HypB-SlyD complex establishes communication between GTP hydrolysis and nickel delivery and provide insight into the role of the HypB-SlyD complex during [NiFe]-hydrogenase biosynthesis.     

Characterization of cobalt(II)-substituted peptide deformylase: function of the metal ion and the catalytic residue Glu-133

Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent ribosome-synthesized polypeptides in eubacteria. PDF represents a novel class of mononuclear iron protein, which utilizes an Fe(2+) ion to catalyze the hydrolysis of an amide bond. This Fe(2+) enzyme is, however, extremely labile, undergoing rapid inactivation upon exposure to molecular oxygen, and is spectroscopically silent. In this work, we have replaced the native Fe(2+) ion with the spectroscopically active Co(2+) ion through overexpression in the presence of Co(2+). Co(2+)-substituted PDF (Co-PDF) has an activity 3-10-fold lower than that of the Fe(2+)-PDF but is highly stable. Steady-state kinetic assays using a series of substrates of varying deformylation rates indicate that Co-PDF has the same substrate specificity as the native enzyme. Co-PDF and Fe-PDF also share the same three-dimensional structure, pH sensitivity, and inhibition pattern by various effector molecules. These results demonstrate that Co-PDF can be used as a stable surrogate of Fe-PDF for biochemical characterization and inhibitor screening. The electronic absorption properties of the Co(2+) ion were utilized as a probe to monitor changes in the enzyme active site as a result of site-directed mutations, inhibitor binding, and changes in pH. Mutation of Glu-133 to an alanine completely abolishes the catalytic activity, whereas mutation to an aspartate results in only approximately 10-fold reduction in activity. Analysis of their absorption spectra under various pH conditions reveals pK(a) values of 6.5 and 5.6 for the metal-bound water in E133A and E133D Co-PDF, respectively, suggesting that the metal ion alone is capable of ionizing the water molecule to generate the catalytic nucleophile, a metal-bound hydroxide. On the other hand, substrate binding to the E133A mutant induces little spectral change, indicating that in the E.S complex the formyl carbonyl oxygen is not coordinated with the metal ion. These results demonstrate that the function of the active-site metal is to activate the water molecule, whereas Glu-133 acts primarily as a general acid, donating a proton to the leaving amide ion during the decomposition of the tetrahedral intermediate.     

Structure and biochemical activities of Escherichia coli MgsA

Bacterial "maintenance of genome stability protein A" (MgsA) and related eukaryotic enzymes play important roles in cellular responses to stalled DNA replication processes. Sequence information identifies MgsA enzymes as members of the clamp loader clade of AAA+ proteins, but structural information defining the family has been limited. Here, the x-ray crystal structure of Escherichia coli MgsA is described, revealing a homotetrameric arrangement for the protein that distinguishes it from other clamp loader clade AAA+ proteins. Each MgsA protomer is composed of three elements as follows: ATP-binding and helical lid domains (conserved among AAA+ proteins) and a tetramerization domain. Although the tetramerization domains bury the greatest amount of surface area in the MgsA oligomer, each of the domains participates in oligomerization to form a highly intertwined quaternary structure. Phosphate is bound at each AAA+ ATP-binding site, but the active sites do not appear to be in a catalytically competent conformation due to displacement of Arg finger residues. E. coli MgsA is also shown to form a complex with the single-stranded DNA-binding protein through co-purification and biochemical studies. MgsA DNA-dependent ATPase activity is inhibited by single-stranded DNA-binding protein. Together, these structural and biochemical observations provide insights into the mechanisms of MgsA family AAA+ proteins.     

Expression, purification, and activity assay of peptide deformylase from Escherichia coli and Staphylococcus aureus

Peptide deformylase (PDF) is considered an attractive target for screening novel antibiotics. The PDF from Escherichia coli and Staphylococcus aureus are representative of the gram-negative species type of PDF (type I PDF) and the gram-positive species type of PDF (type II PDF), respectively. They could be used for screening broad-spectrum antibiotics. Herein, we cloned the def gene by PCR, inserted it into plasmid pET-22b-def, and transformed the plasmid into E. coli BL21 (DE3) cells, then the cells were induced by IPTG to express PDF. E. coli Ni(2+)-PDF was extracted and purified by ion-exchange chromatography and gel filtration chromatography. S. aureus PDFs were extracted and purified using the MagExtractor kit. The nickel form of S. aureus PDF was obtained by adding NiCl(2) to all reagents used for purification. Iron-enriched S. aureus PDF was obtained by adding FeCl(3) to the growth medium for E. coli BL21 (DE3) cells and adding FeCl(3) and catalase to all reagents used for purification. The activities of PDFs were analyzed, compared, and grouped according to the experimental conditions that produced optimal activity, and we used actinonin as an inhibitor of PDF and calculated the IC(50) value. We obtained high expression of E. coli and S. aureus PDF with high activity and stability. The function of PDFs was inhibited by actinonin in a dose-dependent manner. Results may be helpful for future mechanistic investigations of PDF as well as high-throughput screening for other PDF inhibitors.     

Construction and characterization of an Escherichia coli strain genetically engineered for Ni(II) bioaccumulation

An Escherichia coli strain that accumulated Ni(II) was constructed by introducing the nixA gene (coding for a nickel transport system) from Helicobacter pylori into JM109 cells that expressed a glutathione S-transferase-pea metallothionein fusion protein. The resulting strain accumulated 15 micromol of Ni(II) per g (dry weight) from a 10 microM Ni(II) solution, four times the level taken up by JM109 cells. Ni(II) accumulation did not require an energy source, was inhibited by only 50% by 0.1 M NaCl, and occurred over the pH range from 3 to 9.     

Construction and Characterization of an Escherichia coli Strain Genetically Engineered for Ni(II) Bioaccumulation

An Escherichia coli strain that accumulated Ni(II) was constructed by introducing the nixA gene (coding for a nickel transport system) from Helicobacter pylori into JM109 cells that expressed a glutathione S-transferase–pea metallothionein fusion protein. The resulting strain accumulated 15 μmol of Ni(II) per g (dry weight) from a 10 μM Ni(II) solution, four times the level taken up by JM109 cells. Ni(II) accumulation did not require an energy source, was inhibited by only 50% by 0.1 M NaCl, and occurred over the pH range from 3 to 9.     

Effects of coordination of diammineplatinum(II) with DNA on the activities of Escherichia coli DNA polymerase I

The effects of the reaction of cis- and trans-diamminedichloroplatinum(II) with DNA have been measured with regard to DNA synthesis, 3'-5' exonuclease (proofreading), and 5'-3' exonuclease (repair) activities of Escherichia coli DNA polymerase I. Both isomers inhibit DNA synthetic activity of the polymerase through an increase in Km values and a decrease in Vmax values for platinated DNA but not for the nucleoside 5'-triphosphates as the varied substrates. The inhibition is a consequence of lowered binding affinity between platinated DNA and DNA polymerase, and of a platination-induced separation of template and primer strands. Strand separation enhances initial rates of 3'-5' excision of [3H]dCMP from platinated DNA (proofreading), while total excision levels of nucleotides are decreased. In contrast to proofreading activity, the 5'-3' exonuclease activity (repair) discriminates between DNA which had reacted with cis- and with trans-diamminedichloroplatinum(II). While both initial rates and total excision are inhibited for the cis isomer, they are almost not affected for the trans isomer. This differential effect could explain why bacterial growth inhibition requires much higher concentrations of trans- than cis-diamminedichloroplatinum(II).     

A trigonal-prismatic Ag(I) complex and an octahedral Cu(II) complex incorporating with oxamide Ni(II) complex ligand: Syntheses, crystal structures and oxidative nuclease activities

A mononuclear macrocyclic complex Ni^IIL^3a (L^3a = dianion of 2,3-dioxo-5,6:13,14-dibenzo-9,10-cyclohexyl-7,12-bis(methoxycarbonyl)-1,4,8,11-tetraazacyclotetradeca-7,11-diene), which shows high DNA cleavage activity in the presence of H₂O₂, was reported in our previous work. Considering that many systems for natural enzyme-mediated DNA cleavage contain two or more metal active sites, two new trinuclear complexes [Cu(NiL^3a)₂(dca)₂]·2CH₃OH (abbreviated as Cu(NiL^3a)₂) and [Ag(NiL^3a)₂(NO₃)]·2CH₃OH·0.5H₂O (abbreviated as Ag(NiL^3a)₂) were synthesized in this work, where dca is the dicyanamide. The complexes were structurally characterized by single crystal X-ray analysis. The central Cu(II) or Ag(I) atom is linked to two [NiL^3a] ligands by oxamido bridges forming a trinuclear structure. In Cu(NiL^3a)₂, the central Cu(II) ion is in an octahedral coordination geometry. Whereas in Ag(NiL^3a)₂, the central Ag(I) ion is in a rarely reported trigonal-prismatic coordination geometry. The DNA cleavage behavior of the complexes in the presence of H₂O₂ was studied in detail. Comparing with the NiL^3a, the trinuclear complex Ag(NiL^3a)₂ nearly has no ability to cleave DNA, whereas Cu(NiL^3a)₂ is a much better DNA cleavage agent. Cu(NiL^3a)₂ can efficiently convert supercoiled DNA to nicked DNA with a rate constant of 0.074 ± 0.002 min⁻¹ when 40 μM Cu(NiL^3a)₂ and 0.6 mM H₂O₂ are used. The cleavage mechanism between the complex Cu(NiL^3a)₂ and plasmid DNA is likely to involve singlet oxygen as reactive oxygen species. Circular dichroism (CD) and fluorescence spectroscopy indicate that both Cu(NiL^3a)₂ and NiL^3a bind to DNA by a groove binding mode, and the binding between Cu(NiL^3a)₂ and DNA is much stronger than that between NiL^3a and DNA. The present results may provide some information for the design of efficient multinuclear artificial nucleases.     

Effects of polyamines on the activities of Escherichia coli ribonuclease I and II.

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by E. coli ribonuclease I [ribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.23] and ribonuclease II [EC 3.1.4.1] have been studied. The degradation of poly(C) by RNase II was stimulated by spermine and spermidine, while that of poly(A) by RNase II was not affected by polyamines. Under our standard experimental conditions, the breakdown of poly(U) by RNase II was inhibited slightly by polyamines. The stimulatory effect of spermine and spermidine on the breakdown of poly(C) occurred in the absence of monovalent cations but not in the absence of divalent cations. When polyamines were used as a stimulant of RNase II, the ratio of poly(C) degradation to poly(U) degradation was greater in the presence of inhibitors such as poly(G) than in their absence. Although the breakdown of all synthetic polynucleotides by RNase I was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C)greater than poly(A)(see text)poly(U). However, the difference in degree of stimulation among polynucleotides decreased as monovalent cation concentration was increased.     

An Escherichia coli twin-arginine signal peptide switches between helical and unstructured conformations depending on the hydrophobicity of the environment.

The Tat system catalyzes the transport of folded globular proteins across the bacterial plasma membrane and the chloroplast thylakoid. It recognizes cleavable signal peptides containing a critical twin-arginine motif but little is known of the overall structure of these peptides. In this report, we have analyzed the secondary structure of the SufI signal peptide, together with those of two nonfunctional variants in which the region around the twin-arginine, RRQFI, is replaced by KKQFI or RRQAA. Circular dichroism studies show that the SufI peptide exists as an unstructured peptide in aqueous solvent with essentially no stable secondary structure. In membrane-mimetic environments such as SDS micelles or water/trifluoroethanol, however, the peptide adopts a structure containing up to about 40% alpha-helical content. Secondary structure predictions and molecular modelling programs strongly suggest that the helical region begins at, or close to, the twin-arginine motif. Studies on the thermal stability of the helix demonstrate a sharp transition between the unstructured and helical states, suggesting that the peptide exists in one of two distinct states. The two nonfunctional peptides exhibit almost identical spectra and properties to the wild-type SufI peptide, indicating that it is the arginine sidechains, and not their contribution to the helical structure, that are critical in this class of peptide.     

Five different enzymatic activities are associated with the multienzyme complex of fatty acid oxidation from Escherichia coli.

The purified multienzyme complex of fatty acid oxidation from Escherichia coli was found to possess 3-hydroxyacyl-coenzyme A (CoA) epimerase and cis-delta3-trans-delta2-enoyl-CoA isomerase activities in addition to the previously identified enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoactyl-CoA thiolase activities. Evidence is presented in support of the proposed association of all five enzyme activities with one protein which apparently is composed of two types of subunits and which can exist in several aggregated forms. The five component enzymes of the complex were rapidly inactivated by tris(hydroxymethyl)aminomethane, whereas they remained active in the presence of potassium phosphate.     

Structure of the ATP synthase complex (ECF1F0) of Escherichia coli from cryoelectron microscopy

The structural relationship of the catalytic portion (ECF1) of the Escherichia coli F1F0 ATP synthase (ECF1F0) to the intact, membrane-bound complex has been determined by cryoelectron microscopy and image analysis of single, unordered particles. ECF1F0, reconstituted into membrane structures, has been preserved and examined in its native state in a layer of amorphous ice. Side views of the ECF1F0 show the same elongated bilobed and trilobed projection of the ECF1 views shown previously to be normal to the hexagonal projection. The elongated aqueous cavity of the ECF1 is perpendicular to the membrane bilayer profile in the bilobed view. ECF1 is separated from the membrane-embedded F0 by a narrow stalk approximately 40 A long and approximately 25-30 A thick. The F0 part extends from the lipid bilayer by approximately 10 A on the side facing the ECF1. There is no clear extension of the protein on the opposite side of the membrane.     

Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.     

Retention of heme in axial ligand mutants of succinate-ubiquinone xxidoreductase (complex II) from Escherichia coli

Succinate-ubiquinone oxidoreductase (SdhCDAB, complex II) from Escherichia coli is a four-subunit membrane-bound respiratory complex that catalyzes ubiquinone reduction by succinate. In the E. coli enzyme, heme b(556) is ligated between SdhC His(84) and SdhD His(71). Contrary to a previous report (Vibat, C. R. T., Cecchini, G., Nakamura, K., Kita, K., and Gennis, R. B. (1998) Biochemistry 37, 4148-4159), we demonstrate the presence of heme in both SdhC H84L and SdhD H71Q mutants of SdhCDAB. EPR spectroscopy reveals the presence of low spin heme in the SdhC H84L (g(z) = 2.92) mutant and high spin heme in the SdhD H71Q mutant (g = 6.0). The presence of low spin heme in the SdhC H84L mutant suggests that the heme b(556) is able to pick up another ligand from the protein. CO binds to the reduced form of the mutants, indicating that it is able to displace one of the ligands to the low spin heme of the SdhC H84L mutant. The g = 2.92 signal of the SdhC H84L mutant titrates with a redox potential at pH 7.0 (E(m)(,7)) of approximately +15 mV, whereas the g = 6.0 signal of the SdhD H71Q mutant titrates with an E(m)(,7) of approximately -100 mV. The quinone site inhibitor pentachlorophenol perturbs the heme optical spectrum of the wild-type and SdhD H71Q mutant enzymes but not the SdhC H84L mutant. This finding suggests that the latter residue also plays an important role in defining the quinone binding site of the enzyme. The SdhC H84L mutation also results in a significant increase in the K(m) and a decrease in the k(cat) for ubiquinone-1, whereas the SdhD H71Q mutant has little effect on these parameters. Overall, these data indicate that SdhC His(84) has an important role in defining the interaction of SdhCDAB with both quinones and heme b(556).