Search

The spollG operon of Bacillus subtilis codes for a sporulation-specific sigma factor, σE. In vivo expression of the spollG promoter is activated shortly after the onset of sporulation and is dependent on kinA, spo0F, spo0B and spo0A genes. The products of these genes have been shown to participate in a phosphorelay reaction in vitro, culminating in phosphorylation of the transcription factor, Spo0A. The effect of Spo0A phosphorylation on in vitro transcription from the spollG promoter was determined. Aliquots from phosphorelay reactions enhanced spollG promoter activity 10-fold in transcription assays and stimulation of transcription was dependent on Spo0A phosphorylation. Our results provide biochemical evidence that Spo0A and the phosphorelay form a signal transduction pathway which activates spoll gene expression in development. [ABSTRACT FROM AUTHOR]

Rational site-directed mutagenesis and biophysical analyses have been used to explore the thermodynamic stability and catalytic capabilities of organophosphorus hydrolase (OPH) and its genetically modified variants. There are clear trade-offs in the stability of modifications that enhance catalytic activities. For example, the H254R/H257L variant has higher turnover numbers for the chemical warfare agents VX (144 versus 14 s(-1) for the native enzyme (wild type) and VR (Russian VX, 465 versus 12 s(-1) for wild type). These increases are accompanied by a loss in stability in which the total Gibb's free energy for unfolding is 19.6 kcal/mol, which is 5.7 kcal/mol less than that of the wild-type enzyme. X-ray crystallographic studies support biophysical data that suggest amino acid residues near the active site contribute to the chemical and thermal stability through hydrophobic and cation-pi interactions. The cation-pi interactions appear to contribute an additional 7 kcal/mol to the overall global stability of the enzyme. Using rational design, it has been possible to make amino acid changes in this region that restored the stability, yet maintained effective V-agent activities, with turnover numbers of 68 and 36 s(-1) for VX and VR, respectively. This study describes the first rationally designed, stability/activity balance for an OPH enzyme with a legitimate V-agent activity, and its crystal structure.

Organophosphorus hydrolase (OPH) is a bacterial enzyme that hydrolyzes a broad variety of OP neurotoxins, including chemical warfare agents and many widely used pesticides. OPH has extremely high hydrolytic efficiency with different phosphotriester and phophothiolester pesticides (k(cat) = 50-15,000 s(-1)) as well as phosphorofluorates such as DFP and the chemical warfare agents sarin and soman (k(cat) = 50-11,000 s(-1)). In contrast, the enzyme has much lower catalytic capabilities for phosphonothioate neurotoxins such as acephate or the chemical warfare agent VX [O-ethyl S-(2-diisopropyl aminoethyl) methylphosphonothioate] (k(cat) = 0.3-20 s(-1)). Different metal-associated forms of the enzyme have demonstrated varying hydrolytic capabilities for each of the OP neurotoxins, and the activity of OPH (Co2+) is consistently higher than that of OPH (Zn2+) by five- to 20-fold. Protein engineering strategies have exploited these metal-induced catalytic differences, and other slight modifications to the opd gene have resulted in significant enhancement of the rates of detoxification of the thioate pesticides and chemical warfare agents. In order to develop practical applications of OPH, other experiments have focused on improvement of enzyme production, localization, stability, and shelf-life, as well as efficient catalysis of substrates of interest.

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a dimeric, bacterial enzyme that detoxifies many organophosphorus neurotoxins by hydrolyzing a variety of phosphonate bonds. The histidinyl residues at amino acid positions 254 and 257 are located near the bimetallic active site present in each monomer. It has been proposed that these residues influence catalysis by interacting with active site residues and the substrate in the binding pocket. We replaced the histidine at position 254 with arginine (H254R) and the one at position 257 with leucine (H257L) independently to form the single-site-modified enzymes. The double modification was also constructed to incorporate both changes (H254R/H257L). Although native OPH has two metals at each active site (four per dimer), all three of these altered enzymes possessed only two metals per dimer while retaining considerable enzymatic activity for the preferred phosphotriester (P-O bond) substrate, paraoxon (5-100% kcat). The three altered enzymes achieved a 2-30-fold increase in substrate specificity (kcat/Km) for demeton S (P-S bond), an analogue for the chemical warfare agent VX. In contrast, the substrate specificity for diisopropyl fluorophosphonate (P-F bond) was substantially decreased for each of these enzymes. In addition, H257L and H254R/H257L showed an 11- and 18-fold increase, respectively, in specificity for NPPMP, the analogue for the chemical warfare agent soman. These results demonstrate the ability to significantly enhance the specificity of OPH for various substrates by site-specific modifications, and it is suggested that changes in metal requirements may affect these improved catalytic characteristics by enhancing structural flexibility and improving access of larger substrates to the active site, while simultaneously decreasing the catalytic efficiency for smaller substrates.

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.

Phosphotriesterase (EC 3.1.8.1) was immobilized within a polyurethane foam matrix during polymer synthesis using a prepolymer synthesis strategy. In addition to retaining greater than 50% of the enzyme specific activity, numerous benefits were incurred upon immobilization. Orders of magnitude increases in storage and thermal stability (net stabilization energy = 12.5 kJ/mol) were observed without the need for enzyme premodification. The immobilized enzyme system was protease resistant and seemed to display no adverse effects from immobilization, such as an alteration of enzyme function. The organic solvent, dimethyl sulfoxide, also exhibited a stabilizing effect on phosphotriesterase enzyme systems over a range of intermediate concentrations. We attribute these effects in part to direct interaction between the aprotic solvent and metal containing residues present at the enzyme's active site. Our data demonstrate that just 2.5 kg of immobilized enzyme may be sufficient to degrade 30,000 tons of nerve agent in just 1 year.

Sporulation in Bacillus subtilis is dependent on the response regulator Spo0A, which both represses and activates transcription in vitro. The activity of Spo0A is increased by phosphorylation. We previously demonstrated that the phosphorylation increased the ability of Spo0A to stimulate in vivo transcription from the promoter for the spoIIG operon, one of the operons known to be regulated by Spo0A in vivo. In the work reported here we have examined the kinetics of transcription initiation at the spoIIG operon promoter using a single round transcription assay and the kinetics of formation of spoIIG promoter-RNA polymerase complexes using DNase I footprinting. Both the kinetic assays and the footprint assays indicated that the initial binding of the polymerase to the template was not dependent on the presence of Spo0A. The phosphorylated form of Spo0A stimulated the rate of initiation by affecting a step that occurred after the initial interaction of the polymerase with the template. Phosphorylation of Spo0A may stimulate transcription by modifying preinitiation complexes containing the polymerase and the promoter.

The Spo0A transcription factor is responsible for the initiation of sporulation and is active in transcription only after phosphorylation by a specific signal transduction pathway, the phosphorelay. The effect of phosphorylation on the physical properties of Spo0A was determined. Spo0A and Spo0A approximately P both behaved as monomers during Sephacryl chromatography and gel electrophoresis, suggesting that phosphorylation did not modify the oligomerization state of the protein. Trypsin digested Spo0A at a single cleavage site between residues 142 and 143 within a hinge connecting two tightly folded domains. The amino domain retains ability to be phosphorylated by the phosphorelay. The carboxyl domain is active as a DNA-binding protein and retains the sequence specificity of the intact molecule for 0A boxes on the abrB promoter as revealed by footprinting studies. The carboxyl domain stimulated in vitro transcription from the spoIIG promoter 5-fold greater than an equal amount of Spo0A and about half as well as equivalent amounts of Spo0A approximately P. Thus, the unphosphorylated amino domain inhibits the transcription stimulation activity of the carboxyl domain. We suggest that phosphorylation activates transcription regulation functions of Spo0A by modifying the spatial relationships of the amino and carboxyl domains.

A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.