Joseph J. DeFrank

Alteromonas prolidase for organophosphorus G-agent decontamination

Enzymes catalyzing the hydrolysis of highly toxic organophosphorus compounds (OPs) are classified as organophosphorus acid anhydrolases (OPAA; EC 3.1.8.2). Recently, the genes encoding OPAA from two species of Alteromonas were cloned and sequenced. Sequence and biochemical analyses of the cloned genes and enzymes have established Alteromonas OPAAs to be prolidases (E.C. 3.4.13.9), a type of dipeptidase hydrolyzing dipeptides with a prolyl residue in the carboxyl-terminal position (X-Pro). Alteromonas prolidases hydrolyze a broad range of G-type chemical warfare (CW) nerve agents. Efforts to over-produce a prolidase from A. sp.JD6.5 with the goal of developing strategies for long-term storage and decontamination have been successfully achieved. Large-scale production of this G-agent degrading enzyme is now feasible with the availability of an over-producing recombinant cell line. Use of this enzyme for development of a safe and non-corrosive decontamination system is discussed.

Enzymatic hydrolysis of the chemical warfare agent VX and its neurotoxic analogues by organophosphorus hydrolase

Organophosphorus hydrolase (OPH) is a bacterial enzyme that hydrolyzes a variety of organophosphorus (OP) neurotoxins, including many widely used pesticides and chemical warfare agents containing P-O, P-F, P-CN and P-S bonds. It has extremely high efficiency in hydrolysis of many different phosphotriester and phosphothiolester pesticides (P-O bond) such as paraoxon (kcat3800s−1) and coumaphos (kcat = 800s−1) or phosphonate (P-F) neurotoxins such as DFP (kcat = 350s−1) and the chemical warfare agent sarin (kcat = 56s−1). In contrast, the enzyme has much lower catalytic capabilities for phosphonothioate neurotoxins such as acephate (kcat = 2.8 s−1) or the chemical warfare agent VX [O-ethyl S-(2-diisopropyl-aminoethyl) methylphosphonothioate] (kcat = 0.3s−1). This lower specificity for VX and its analogues are reflected by the specificity constants (kcat/Km values) for VX = 0.75 × 103 M−1 s−1 compared to 5.5 × 107M−1 s−1 for paraoxon. Different metal-associated forms of the enzyme demonstrated significantly ...

Screening of halophilic bacteria and Alteromonas species for organophosphorus hydrolyzing enzyme activity.

Previously, a G-type nerve agent degrading enzyme activity was found in a halophilic bacterial isolate designated JD6.5. This organism was tentatively identified as an unknown species of the genus Alteromonas. In order to determine whether this type of enzyme activity was common in other species of Alteromonas, a screening program was initiated. A number of Alteromonas species and five halophilic bacterial isolates were cultured and their crude cell extracts screened for hydrolytic activity against several organophosphorus chemical agents and other related compounds. The samples were also screened for cross-reactivity with a monoclonal antibody raised against the purified enzyme from JD6.5 and for hybridization with a DNA probe based on its N-terminal amino acid sequence A wide spectrum of activities and reactivities were seen, suggesting a significant heterogeneity between the functionally similar enzymes that are present in these bacterial species. Enzymes of the type described here have considerable potential for the decontamination and demilitarization of chemical warfare agents.

Substrate and stereochemical specificity of the organophosphorus acid anhydrolase from Alteromonas sp. JD6.5 toward p-nitrophenyl phosphotriesters

The enzyme OPAA hydrolyzes p-nitrophenyl phosphotriesters bearing substituents at the phosphorus center ranging in size from methyl to phenyl. The enzyme exhibits stereoselectivity toward the hydrolysis of chiral substrates with a preference for the Sp enantiomer.

Phosphofluoridates: Biological Activity and Biodegradation

The combination of phosphorus and fluorine has resulted in a variety of compounds that are unique in their physical and chemical properties as well as being some of the most toxic materials produced by man. A review of the chemistry and enzymatic basis for the toxicology of these compounds is presented. Catalytic enzymes that use these compounds as substrates and their potential applications are also reviewed.

Hydrolysis of Organophosphorus Compounds by Bacterial Prolidases

Numerous organophosphorus (OP) compounds of importance in agriculture, medicine, military defense, and research have been shown to be potent inhibitors of cholinesterases and other enzymes with active serine residues in their active sites. Enzymes that catalyze the hydrolysis of OP compounds have been under investigation for over 50 years. These enzymes result in the detoxification of a variety of these highly toxic compounds, including chemical warfare (CW) nerve agents and pesticides. For military operations, enzyme-based decontamination systems offer considerable potential for replacing current materials that are toxic, highly corrosive, flammable, and a danger to the environment.

Infrared Reflection-Absorption Spectroscopy and Polarization-Modulated Infrared Reflection-Absorption Spectroscopy Studies of the Organophosphorus Acid Anhydrolase Langmuir Monolayer

The secondary structure of the organophosphorus acid anhydrolase (OPAA) Langmuir monolayer in the absence and presence of diisopropylfluorophosphate (DFP) in the subphase was studied by infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated IRRAS (PM-IRRAS). The results of both the IRRAS and the PM-IRRAS indicated that the alpha-helix and the beta-sheet conformations in OPAA were parallel to the air-water interface at a surface pressure of 0 mN.m-1 in the absence of DFP in the subphase. When the surface pressure increased, the alpha-helix and the beta-sheet conformations became tilted. When DFP was added to the subphase at a concentration of 1.1 x 10(-5) M, the alpha-helix conformation of OPAA was still parallel to the air-water interface, whereas the beta-sheet conformation was perpendicular at 0 mN.m-1. The orientations of both the alpha-helix and the beta-sheet conformations did not change with the increase of surface pressure. The shape of OPAA molecules is supposed to be elliptic, and the long axis of OPAA was parallel to the air-water interface in the absence of DFP in the subphase, whereas the long axis became perpendicular in the presence of DFP. This result explains the decrease of the limiting molecular area of the OPAA Langmuir monolayer when DFP was dissolved in the subphase.

Langmuir and Langmuir-Blodgett films of organophosphorus acid anhydrolase

In this paper, we describe the preparation and characterization of Langmuir and Langmuir-Blodgett (LB) monolayers of the enzyme organophosphorus acid anhydrolase (OPAA). Langmuir films of OPAA were characterized on different subphases, such as phosphate, ammonium carbonate, and bis-tris-propane buffers. Monolayers at the air-water interface were characterized by measuring the surface pressure and surface potential-area isotherms. In situ UV-vis absorption spectra were also recorded from the Langmuir monolayers. The enzyme activity at the air-water interface was tested by the addition of diisopropylfluorophosphate (DFP) to the subphase. LB films of OPAA were transferred to mica substrates to be studied by atomic force microscopy. Finally, a one-layer LB film of OPAA labeled with a fluorescent probe, fluorescein isothiocyanate (FITC), was deposited onto a quartz slide to be tested as sensor for DFP. The clear, pronounced response and the stability of the LB film as a DFP sensor show the potential of this system as a biosensor.

Interaction between organophosphorous hydrolase and paraoxon studied by surface chemistry in situ at air-water interface

Subphase conditions have been optimized to obtain stable organophosphorous hydrolase (OPH-EC 3.1.8.1) as Langmuir films. The Langmuir film was characterized by surface pressure and surface potential-area isotherms and UV-Vis spectroscopy in situ. The interaction of an organophosphorous compound, namely Paraoxon, with the OPH film was investigated for various surface pressures. The stability of the monolayer and the evidence of the enzyme activity at air-water interface support the use of enzyme LB films as biosensor.

Microbial Degradation of Agent Orange and Mustard Related Compounds

We live in a highly industrialized society where chemicals play a major role in enhancing industrial and agricultural productivity, in providing physical comforts to our day to day life, as well as in military operations as explosives, ammunitions and agents of chemical warfare. Highly toxic chemicals such as nitrogen and sulfur mustards have been stockpiled for military purposes, and as international treaties leading to their gradual destruction are signed, appropriate technologies will have to be developed for large-scale destruction of such stockpiled chemicals in a cost effective and environmentally safe way. Bioremediation, which uses biological agents mostly in the form of microorganisms or products derived from them, is considered to be highly desirable if appropriate microorganisms can be developed for the rapid degradation of the toxic chemicals. Unfortunately, most toxic chemicals are synthetic and have been introduced in the environment only during the last few decades. Many of these compounds are highly persistent in nature, because natural microorganisms have not had enough time to evolve the appropriate genetic capability to mineralize such compounds. In this article, we briefly consider two types of chemicals that have found military use, viz. Agent Orange used as a defoliant in the Vietnam war and sulfur mustard which is a toxic vesicant stockpiled for intended use as a chemical warfare agent.