Reconstruction of the active site of a bacterial phosphotriesterase for the catalytic hydrolysis and detoxification of organophosphate nerve agents

The bacterial phosphotriesterase (PTE), originally purified from the bacterium Pseudomonas diminuta, catalyzes the hydrolysis of the organophosphate insecticide paraoxon at a rate near the diffusion-controlled limit. The protein has been crystallized and the three-dimensional structure determined to high resolution. The protein adopts a (β/α)8-barrel structural fold and the active site is dominated by a binuclear metal center with a bridging hydroxide that is used for direct nucleophilic attack on the phosphorus center of the substrate. The wild-type enzyme is stereoselective for the hydrolysis of chiral organophosphate substrates. For example, the wild-type enzyme preferentially hydrolyzes the SP-enantiomer of methyl phenyl p-nitrophenyl phosphate by a factor of 93:1. The mutation of Gly-60 to alanine (G60A) enhances the preference for hydrolysis of the SP-enantiomer to a factor of 13,000:1 by reducing the rate of hydrolysis of the RP-enantiomer. The stereoselectivity of PTE can be reversed by mutation of three residues within the active site (I106G/F132G/H257Y) and now the RP-enantiomer is preferentially hydrolyzed by a factor of 118:1. Therefore, the stereoselectivity can be changed by more than four orders-of-magnitude by mutation of only four amino acids changes in the active site and thus PTE variants can be used to resolve racemic mixtures of chiral organophosphate esters. PTE was demonstrated to catalyze the hydrolysis of the organophosphate nerve agents tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VX, and VR. Screening of a small library of active site mutants identified one mutant (H257Y/L303T) that was particularly efficient toward the hydrolysis of the G-agents (in collaboration with Dr. Steve Harvey). The YT mutant of PTE hydrolyzed GB with a kcat of 3.1 x 104 min-1. This mutant, at a dose of 1 mg/Kg, was shown to significantly protect guinea pigs from exposure to sarin. The LD50 increased by more than a factor of 65 (in collaboration with Dr. Douglas Cerasoli). Coating of the surface of the YT variant with a thin ultrahydrophilic semipermeable poly(carboxybetaine) polymer enhanced protein stability, reduced immunological complications and protected rodents from repeated exposures to sarin over a period of 1-week (in collaboration with Dr. Shaoyi Jiang). From a 30,000 member mutant library, multiple variants were identified for the catalytic hydrolysis of VX and VR. The best mutants identified to date for the hydrolysis of (SP)-VX and (SP)-VR were enhanced more than 10,000-fold, relative to the wild-type PTE (in collaboration with Dr. S. Harvey). These results demonstrate that the active site of PTE can be readily manipulated to engineer protein variants with enhanced catalytic properties for hydrolysis of highly toxic organophosphate nerve agents

Investigations on the Mechanism of Allosteric Activtion of Rabbit Muscle Glycogen Phosphorylase b by AMP

Much work has been carried out on glycogen phosphorylase over the last seventy years. Interest has persisted due not only to the usefulness of phosphorylase as a model system of allostery, but also due to the connection to the disease state in type II diabetes. The bulk of research consists of structural studies utilizing the wild-type enzyme from rabbit muscle. In this study we have employed linkage analysis in combination with structural perturbations via site-directed mutagenesis to test kinetic models of activation of phosphorylase b by AMP, and to examine the roles of the N-terminus, the acidic patch, ?-helix 1 and the 280?s loop in activation by AMP. Experiments have been carried out on purified glycogen phosphorylase b variants to determine the effects of perturbations in vitro. The kinetic models of activation by AMP are found to be a relatively accurate description of kinetic behavior of wild-type phosphorylase b, but are found to be technically incorrect with respect to the absolute requirements of two equivalents of AMP to be bound prior to catalysis. Phosphorylase b demonstrates activity in the absence of AMP, though only at high concentrations of phosphate, and a hybrid phosphorylase b with only a single functional AMP binding sight shows slight activation. The truncate ?2-17 shows weakened binding to AMP and phosphate in the apo enzyme, but maintains activation by AMP to an affinity similar to that of wild-type, indicating that the N-terminus is not required for activation by AMP, but has a role in establishing the affinity for both AMP and phosphate in the apo enzyme. Perturbations of the acidic patch indicate that interactions between the acidic patch and the N-terminus enhance the affinities in the apo enzyme, suggesting that the structures of the N-terminus at the acidic patch may represent an active form of the enzyme. ?-helix 1 is found to have a role in homotropic cooperativity in phosphorylase b, but not in heterotropic activation by AMP, while the 280?s loop is confirmed to have a role in the heterotropic coupling between AMP and phosphate. Based on the findings in this study an alternate structural model of activation by AMP involving ?-helix 8 is proposed

Transition State Analysis of the Reaction Catalyzed by the Phosphotriesterase from Sphingiobium sp. TCM1

Organophosphorus flame retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the hydrolysis of the unactivated substituents is not known. To interrogate the catalytic hydrolysis mechanism of Sb-PTE, the pH dependence of the reaction and the effects of changing the solvent viscosity were determined. These experiments were complemented by measurement of the primary and secondary 18-oxygen isotope effects on substrate hydrolysis and a determination of the effects of changing the pKa of the leaving group on the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a single group must be ionized for nucleophilic attack and that a separate general acid is not involved in protonation of the leaving group. The Brønsted analysis and the heavy atom kinetic isotope effects are consistent with an early associative transition state with subsequent proton transfers not being rate limiting. A novel binding mode of the substrate to the binuclear metal center and a catalytic mechanism are proposed to explain the unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of organophosphate substrates

Imaging muscle as a potential biomarker of denervation in motor neuron disease

Objective To assess clinical, electrophysiological and whole-body muscle MRI measurements of progression in patients with motor neuron disease (MND), as tools for future clinical trials, and to probe pathophysiological mechanisms in vivo. Methods A prospective longitudinal observational clinico-electrophysiological and radiological cohort study was performed. Twenty-nine MND patients and 22 age and gender-matched healthy controls were assessed with clinical measures, electrophysiological motor unit number index (MUNIX) and T2-weighted whole-body muscle MRI, at first clinic presentation and four months later. Between-group differences and associations were assessed using age and gender-adjusted multivariable regression models. Within-subject longitudinal changes were assessed using paired t-tests. Patterns of disease spread were modelled using mixed-effects multivariable regression, assessing associations between muscle relative T2 signal and anatomical adjacency to site of clinical onset. Results MND patients had 30% higher relative T2 muscle signal than controls at baseline (all-regions mean, 95%CI 15%, 45%, p<0.001). Higher T2 signal was associated with greater overall disability (coefficient -0.009, 95%CI -0.017, -0.001, p=0.023), and with clinical weakness and lower MUNIX in multiple individual muscles. Relative T2 signal in bilateral tibialis anterior increased over four months in MND patients (right: 10.2%, 95%CI 2.0%, 18.4%, p=0.017; left: 14.1%, 95%CI 3.4%, 24.9%, p=0.013). Anatomically contiguous disease spread on MRI was not apparent in this model. Conclusions Whole-body muscle MRI offers a new approach to objective assessment of denervation over short timescales in MND, and enables investigation of patterns of disease spread in vivo. Muscles inaccessible to conventional clinical and electrophysiological assessment may be investigated using this methodology

Maritime Operations and Emergency Preparedness in the Arctic–Competence Standards for Search and Rescue Operations Contingencies in Polar Waters

Emergencies on large passenger ships in the remote High North may lead to a mass rescue operation with a heavy strain on the emergency preparedness systems of the Arctic countries. This study focuses on the need for competencies related to large-scale Search and Rescue operations (SAR operations) amongst the shipping companies, vessels and governments involved. A SAR operation is the activity related to finding and rescuing people in distress. Several international standards, in particular the conventions by the International Maritime Organization (IMO), provide direction for education and training of seafarers and rescue staff. This study elaborates on the operational competence requirements for key personnel involved in large scale SAR operations. Findings from real SAR incidents and exercises provide in-depth understanding on the operational challenges. The chapter gives directions for competence programs, beyond obligatory international standards, and recommendations for further research

Space Telescope and Optical Reverberation Mapping Project. XII. Broad-line Region Modeling of NGC 5548

We present geometric and dynamical modeling of the broad line region (BLR) for the multi-wavelength reverberation mapping campaign focused on NGC 5548 in 2014. The data set includes photometric and spectroscopic monitoring in the optical and ultraviolet, covering the Hβ, C iv, and Ly broad emission lines. We find an extended disk-like Hβ BLR with a mixture of near-circular and outflowing gas trajectories, while the C iv and Ly BLRs are much less extended and resemble shell-like structures. There is clear radial structure in the BLR, with C iv and Ly emission arising at smaller radii than the Hβ emission. Using the three lines, we make three independent black hole mass measurements, all of which are consistent. Combining these results gives a joint inference of . We examine the effect of using the V band instead of the UV continuum light curve on the results and find a size difference that is consistent with the measured UV-optical time lag, but the other structural and kinematic parameters remain unchanged, suggesting that the V band is a suitable proxy for the ionizing continuum when exploring the BLR structure and kinematics. Finally, we compare the Hβ results to similar models of data obtained in 2008 when the active galactic nucleus was at a lower luminosity state. We find that the size of the emitting region increased during this time period, but the geometry and black hole mass remained unchanged, which confirms that the BLR kinematics suitably gauge the gravitational field of the central black hole

Space Telescope and Optical Reverberation Mapping Project. VII. Understanding the Ultraviolet Anomaly in NGC 5548 with X-Ray Spectroscopy

During the Space Telescope and Optical Reverberation Mapping Project observations of NGC 5548, the continuum and emission-line variability became decorrelated during the second half of the six-month-long observing campaign. Here we present Swift and Chandra X-ray spectra of NGC 5548 obtained as part of the campaign. The Swift spectra show that excess flux (relative to a power-law continuum) in the soft X-ray band appears before the start of the anomalous emission-line behavior, peaks during the period of the anomaly, and then declines. This is a model-independent result suggesting that the soft excess is related to the anomaly. We divide the Swift data into on- and off-anomaly spectra to characterize the soft excess via spectral fitting. The cause of the spectral differences is likely due to a change in the intrinsic spectrum rather than to variable obscuration or partial covering. The Chandra spectra have lower signal-to-noise ratios, but are consistent with the Swift data. Our preferred model of the soft excess is emission from an optically thick, warm Comptonizing corona, the effective optical depth of which increases during the anomaly. This model simultaneously explains all three observations: the UV emission-line flux decrease, the soft-excess increase, and the emission-line anomaly

Space Telescope and Optical Reverberation Mapping Project. V. Optical Spectroscopic Campaign and Emission-line Analysis for NGC 5548

We present the results of an optical spectroscopic monitoring program targeting NGC 5548 as part of a larger multiwavelength reverberation mapping campaign. The campaign spanned 6 months and achieved an almost daily cadence with observations from five ground-based telescopes. The Hβ and He ii λ4686 broad emission-line light curves lag that of the 5100 +-optical continuum by 4.17+0.36-0.36 and 0.79+0.35-0.34 days, respectively. The Hβ lag relative to the 1158 ultraviolet continuum light curve measured by the Hubble Space Telescope is ∼50% longer than that measured against the optical continuum, and the lag difference is consistent with the observed lag between the optical and ultraviolet continua. This suggests that the characteristic radius of the broad-line region is ∼50% larger than the value inferred from optical data alone. We also measured velocity-resolved emission-line lags for Hβ and found a complex velocity-lag structure with shorter lags in the line wings, indicative of a broad-line region dominated by Keplerian motion. The responses of both the Hβ and He ii emission lines to the driving continuum changed significantly halfway through the campaign, a phenomenon also observed for C iv, Lyα, He ii(+O iii]), and Si iv(+O iv]) during the same monitoring period. Finally, given the optical luminosity of NGC 5548 during our campaign, the measured Hβ lag is a factor of five shorter than the expected value implied by the R BLR-L AGN relation based on the past behavior of NGC 5548

The Berkeley sample of Type II supernovae: BVRI light curves and spectroscopy of 55 SNe II

In this work, BVRI light curves of 55 Type II supernovae (SNe II) from the Lick Observatory Supernova Search programme obtained with the Katzman Automatic Imaging Telescope and the 1 m Nickel telescope from 2006 to 2018 are presented. Additionally, more than 150 spectra gathered with the 3 m Shane telescope are published. We conduct an analyse of the peak absolute magnitudes, decline rates, and time durations of different phases of the light and colour curves. Typically, our light curves are sampled with a median cadence of 5.5 d for a total of 5093 photometric points. In average, V-band plateau declines with a rate of 1.29 mag (100 d)−1, which is consistent with previously published samples. For each band, the plateau slope correlates with the plateau length and the absolute peak magnitude: SNe II with steeper decline have shorter plateau duration and are brighter. A time-evolution analysis of spectral lines in term of velocities and pseudo-equivalent widths is also presented in this paper. Our spectroscopic sample ranges between 1 and 200 d post-explosion and has a median ejecta expansion velocity at 50 d post-explosion of 6500 km s−1 (H α line) and a standard dispersion of 2000 km s−1. Nebular spectra are in good agreement with theoretical models using a progenitor star having a mass <16M⊙. All the data are available to the community and will help to understand SN II diversity better, and therefore to improve their utility as cosmological distance indicators