Janet K. Grimsley

Dramatically stabilized phosphotriesterase—polymers for nerve agent degradation

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 enzymes 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.

Rational design of organophosphorus hydrolase for altered substrate specificities

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.

Improved pharmacokinetics and immunogenicity profile of organophosphorus hydrolase by chemical modification with polyethylene glycol

A catalytic bioscavenger with broad substrate specificity for the therapeutic and prophylactic defense against recognized chemical threat agents has been a long standing objective of civilian and military research. A catalytic bioscavenger utilizing the bacterial enzyme organophosphorus hydrolase (OPH) is characterized in these studies, and has potential application for both military and civilian personnel in threat scenarios involving either nerve agents or OP pesticides. The present study examines the effects of PEGylation on the biochemical and pharmacological characteristics of OPH. The enzyme was conjugated with linear and branched methyl-PEO(n)-NHS esters of relatively small molecular mass from 333 to 2420Da. PEGylated OPH displayed a decreased maximal catalytic rate, though substantial activity was maintained against two tested substrates: up to 30% with paraoxon and up to 50-60% with demeton-S. The thermostability of the PEGylated enzymes ranged between 60 and 64 degrees C, compared to the unmodified OPH, which is approximately 67 degrees C. The enzyme conjugates revealed a significant improvement of pharmacokinetic properties in animal studies. The clearance from a guinea pigs blood stream significantly decreased relative to unmodified OPH, resulting in an increase of residence time and systemic availability. Evaluation of the humoral immune response indicated that the branched PEG-OPH conjugate significantly reduced production of anti-OPH antibodies, compared to the unmodified enzyme. The OPH-PEG conjugates with improved pharmacokinetic and immunogenicity properties, considerable catalytic activity and thermal stability provide a new opportunity for the in vivo detoxification of the neurotoxic OP compounds.

Neurotoxic Organophosphate Degradation with Polyvinyl Alcohol Gel-Immobilized Microbial Cells

A genetically engineered strain of Escherichia coli that expresses organophosphorus hydrolase (OPH) was immobilized in a polyvinyl alcohol (PVA) cryogel to form a porous biocatalyst that successfully degrades organophosphorus (OP) neurotoxins. The impacts of both diffusion and reaction on biocatalyst efficiency were determined to enable prediction and optimization of the biocatalyst performance. The kinetic rate parameters and activation energies of pure OPH, free cell suspensions, and the immobilized cell biocatalyst were compared. Diffusion was a determining factor for paraoxon hydrolysis because of the very rapid OPH kinetics for its model substrate. Both the paraoxon diffusion through the PVA matrix and the diffusion associated with microbial transport of paraoxon were shown to impact the biocatalyst reaction. However, the enhancement in storage stability resulting from diffusional limitations provides an advantage to diffusion-limited operation. This research may serve as a guide to define the influe...

Active Site Modifications of Organophosphorus Hydrolase for Improved Detoxification of Organophosphorus Neurotoxins

Amino acid substitutions within the active site of the dimeric metalloenzyme Organophosphorus Hydrolase (OPH) result in a striking enhancement in the hydrolysis of certain chemical warfare agents and their analogues. These changes alter the metal content of the enzyme and we suggest that changes in metal requirements improve the catalytic characteristics by allowing greater structural flexibility and access of larger substrates to the active site. Crystallographic and three-dimensional modeling analyses have suggested that removing steric hindrances in the vicinity of the binding pocket could further enhance the effectiveness of OPH to hydrolyze VX. These studies also suggest that the hydrogen-bonding network supplying support and stability to the active site deserve a critical analysis for further catalytic improvements. The broad substrate specificity and hydrolytic efficiency of OPH and the ability to genetically engineer the enzyme for specific target organophosphate neurotoxins have provided realistic OPHbased technologies for detoxification of these compounds, including enzyme immobilization on various matrices, discriminative detection, and clinical therapy. A series of optimized enzymes for individual substrates can be envisioned that would maximize degradative activity under a particular environmental situation. The capacity for further improvement is remarkable, and the opportunity for a variety of biotechnological applications is quite pronounced.

Ancillary Function of Housekeeping Enzymes: Fortuitous Degradation of Environmental Contaminants

Most of the environmental contamination of greatest concern around the world today involves xenobiotic materials which have only been produced or used in industrial/agricultural production over the past several decades. These include various organic chemical solvents, heavy metals, neurotoxic pesticides, halogenated aromatic compounds, explosives and carcinogenic industrial chemicals which may or may not have natural counterparts. These have been synthesized with increasing diversity and volume over the last fifty to seventy years. Although their introduction into the environment is lamentable, and the effects they can have on the environment devastating, many different types of microbial systems have already acquired the ability to chemically modify many of these compounds. The likelihood that totally new enzymes with specific detoxification activities could emerge in such a brief period of time is problematic. It is much more likely that enzymes already possessed by microbial communities, possibly used for maintenance functions, can bee recruited to address the new challenge. For example, FMN oxidoreductases may serve to reduce trinitrotoluene and a human lipid-phosphoesterase or a bacterial peptidase can degrade organophosphate pesticides. Five categories of reactions wherein housekeeping enzymes may be able to catalyze ancillary reactions involving previously unknown chemicals include 1) general oxidoreductases, 2) denitrases, 3) phosphoesterases, 4) ring cleavage enzymes and 5) metal-sequestering proteins. Once a potential metabolic route is established, directed evolution based on random mutation or substitutional recombination may serve to enhance activity if a competitive metabolic edge could result.