Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k(cat) than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.     

Functional effects of amino acid substitutions within the large binding pocket of the phosphotriesterase OpdA from Agrobacterium sp. P230

The phosphotriesterase OpdA from Agrobacterium sp. P230 has about 10-fold higher activity for dimethyl organophosphate (OP) insecticides, than its homologue from Flavobacterium sp. ATCC27551, organophosphate hydrolase (OPH). OpdA shows about 10% amino acid sequence divergence from OPH and also has a 20 residue C-terminal extension. Here we show that the difference in kinetics is largely explained by just two amino acid differences between the two proteins. A truncated form of OpdA demonstrated that the C-terminal extension has no effect on its preference for dimethyl organophosphate substrates. Chimeric proteins of OPH and OpdA were then analysed to show that replacement of a central region of OpdA sequence, which encodes the residues in the large subsite of the active site, with the homologous region in OPH decreased the activity of OpdA towards dimethyl OPs, to values close to those for OPH. Site-directed mutagenesis in this region identified two differences between the proteins, Y257H and F272L (with the OpdA residues first) as being responsible for this reduction. These two differences were also responsible for the increased activity of OpdA towards the diisopropyl organophosphate, diisopropyl fluorophosphate, relative to OPH. Molecular modelling of triethyl phosphate in the active site of OpdA confirmed a reduction in the size of the large subsite relative to OPH.     

Organophosphate Hydrolase in Brevundimonas diminuta Is Targeted to the Periplasmic Face of the Inner Membrane by the Twin Arginine Translocation Pathway

A twin arginine translocation (Tat) motif, involved in transport of folded proteins across the inner membrane, was identified in the signal peptide of the membrane-associated organophosphate hydrolase (OPH) of Brevundimonas diminuta. Expression of the precursor form of OPH carrying a C-terminal His tag in an opd-negative background and subsequent immunoblotting with anti-His antibodies showed that only the mature form of OPH associated with the membrane and that the precursor form of OPH was entirely found in the cytoplasm. When OPH was expressed without the signal peptide, most of it remained in the cytoplasm, where it was apparently correctly folded and showed activity comparable to that of the membrane-associated OPH encoded by the wild-type opd gene. Amino acid substitutions in the invariant arginine residues of the Tat signal peptide affected both the processing and localization of OPH, confirming a critical role for the Tat system in membrane targeting of OPH in B. diminuta. The localization of OPH to the periplasmic face of the inner membrane in B. diminuta was demonstrated by proteinase K treatment of spheroplasts and also by fluorescence-activated cell sorting analysis of cells expressing OPH-green fluorescent protein fusions with and without an SsrA tag that targets cytoplasmic proteins to the ClpXP protease.Bacterial organophosphate hydrolases (OPH), also known as phosphotriesterases, have been shown to hydrolyze a structurally diverse group of phosphotriesters used as insecticides and chemical warfare agents (26, 37). The genetic information required to encode these dimeric metalloenzymes is highly conserved and often located on plasmids known as organophosphate-degrading (opd) plasmids (27). Among the opd plasmids, pPDL2 (40 kb), isolated from Flavobacterium sp. strain ATCC27551, and pCMS1 (66 kb), isolated from Brevundimonas diminuta (formerly Pseudomonas diminuta), are well characterized (27). In these two indigenous plasmids, a 7-kb region that includes the 1.5-kb organophosphate-degrading (opd) gene is highly conserved and has the features of a complex transposon (38).OPH has been crystallized from a number of sources and has been shown to be a dimeric metalloenzyme with zinc at its catalytic center (1, 2, 28). In Flavobacterium and B. diminuta, the protein has been shown to be membrane associated, and a 29-amino-acid-long signal peptide found in its precursor form has been deduced to be responsible for membrane targeting (24, 25, 36). A similar signal sequence is also encoded in opd genes identified in Agrobacterium radiobacter (15) and Sphingomonas sp. strain JK1 (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"ACD85809","term_id":"189303769","term_text":"ACD85809"}}ACD85809). While the conservation of a signal peptide in this group of organophosphate hydrolases has been recognized for some time, its biological role and its precise involvement in the membrane localization of OPH have not been investigated. In this study, we expressed OPH with a C-terminal His tag in opd-negative mutants of B. diminuta and established a system to differentiate and localize precursor and mature forms of OPH. We have used this system, together with mutagenesis of the signal peptide-encoding region of the opd gene, to demonstrate that membrane targeting of OPH is dependent on the twin arginine translocation (Tat) protein secretion pathway, which facilitates localization of OPH to the periplasmic face of the inner membrane.     

Biodegradation of organophosphorus insecticides by two organophosphorus hydrolase genes (opdA and opdE) from isolated Leuconostoc mesenteroides WCP307 of kimchi origin

This study is aimed to reveal the molecular incidence of organophosphorus insecticides degradation during the fermentation of Korean food yeulmu-mulkimchi. To this end, two opdA and opdE which consist of 930 and 894 bp that encode 309 and 297 amino acids, respectively, were cloned from the Leuconostoc mesenteroides WCP307 strain that was isolated from chlorpyrifos (CP) impregnated kimchi. The Escherichia coli that harbored the opdA and opdE genes depleted a CP concentration of 72% and 83%, respectively, in an M9 medium after 6 days. The OpdA and OpdE enzymes molecular weights were estimated to be approximately 35 and 33 kDa and showed optimal activities at 30 °C with a pH of 7.0 and 6.0, respectively. However, the mutated OpdA (Ser128 → Ala128) and OpdE (Ser129 → Ala129) enzymes had no activities on OP insecticides and ρ-nitrophenyl butyrate substrates. In addition, the OpdA and OpdE enzymes showed profound catalytic activities against cadusafos, comnaphos, diazinon, dyfonate, ethoprophos, fenamiphos, methylparathion, and parathion insecticides. Therefore, it is assumed that OpdA and OpdE enzymes detoxified the pesticides contaminated kimchi composition like Chinese cabbages during fermentation. Furthermore, the OpdA and OpdE enzymes augmented the diversity of new LAB-opd enzymes group in nature.     

Growth of Escherichia coli Coexpressing Phosphotriesterase and Glycerophosphodiester Phosphodiesterase, Using Paraoxon as the Sole Phosphorus Source

Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The Escherichia coli strain DH10B expressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source. Enterobacter aerogenes is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled E. coli to grow when dimethyl phosphate is the sole phosphorus source. E. coli coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled E. coli to use phosphotriesters as a sole source of phosphorus.     

Immobilization of organophosphohydrolase OpdA from Agrobacterium radiobacter by overproduction at the surface of polyester inclusions inside engineered Escherichia coli

Organophosphorus pesticides (OP) are highly toxic and are widely used as insecticides. Bacterial organophosphohydrolases which hydrolyze a variety of OPs have been considered for the clean-up of polluted environments. This study describes the engineering of Escherichia coli towards the overproduction of the organophosphohydrolase (OpdA) from Agrobacterium radiobacter at the surface of polyester inclusions. The OpdA was N-terminally fused via a designed linker region to the C-terminus of polyester inclusion-forming enzyme PhaC of Ralstonia eutropha. The PhaC-L-OpdA fusion protein was overproduced by using the strong T7 promoter and when coexpressed with genes phaA (encoding β-ketothiolase) and phaB (encoding acetoacetyl-CoA reductase) from R. eutropha this led to formation of polyester inclusions abundantly displaying OpdA. These OpdA beads showed organophosphohydrolase activity of 1,840 U/g wet polyester beads or 4,412 U/g protein. Steady state kinetics revealed that when compared with free OpdA the k(cat) (s(-1)) of 139 of immobilized OpdA was reduced by about 16.5-fold while the K(M) (M) of 2.5 × 10(-4) was increased by 1.6-fold. The immobilized OpdA showed increased temperature stability. Moreover, the stability of OpdA immobilized to polyester beads was assessed by incubating OpdA beads at 25°C for up to 11 days and no significant loss in enzyme activity was detected. The application performance of the OpdA beads with respect to hydrolysis of OPs in contaminated environments was demonstrated in wool scour spiked with fluorescent coumaphos. This study demonstrated a new strategy toward the efficient recombinant production of immobilized organophosphohydrolase, the OpdA, suitable for bioremediation applications.     

Identification of opdA, a Gene Involved in Biodegradation of the Endocrine Disrupter Octylphenol

Octylphenol (OP) is an estrogenic detergent breakdown product. Structurally similar nonylphenols are transformed via type II ispo substitution, resulting in the production of hydroquinone and removal of the branched side chain. Nothing is known, however, about the gene(s) encoding this activity. We report here on our efforts to clone the gene(s) encoding OP degradation activity from Sphingomonas sp. strain PWE1, which we isolated for its ability to grow on OP. A fosmid library of PWE1 DNA yielded a single clone, aew4H12, which accumulated a brown polymerization product in the presence of OP. Sequence analysis of loss-of-function transposon mutants of aew4H12 revealed a single open reading frame, opdA, that conferred OP degradation activity. Escherichia coli subclones expressing opdA caused OP disappearance, with the concomitant production of hydroquinone and 2,4,4-trimethyl-1-pentene as well as small amounts of 2,4,4-trimethyl-2-pentanol. These metabolites are consistent with a type II ipso substitution reaction, the same mechanism described for nonylphenol biodegradation in other sphingomonads. Based on opdA's sequence homology to a unique group of putative flavin monooxygenases and the recovery of hydroxylated OP intermediates from E. coli expressing opdA, we conclude that this gene encodes the observed type II ipso substitution activity responsible for the initial step in OP biodegradation.     

Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on Escherichia coli Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity

Surface display of the active proteins on living cells has enormous potential in the degradation of numerous toxic compounds. Here, we report the codisplay of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (GFP) on the cell surface of Escherichia coli by use of the truncated ice nucleation protein (INPNC) and Lpp-OmpA fusion systems. The surface localization of both INPNC-OPH and Lpp-OmpA-GFP was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. Anchorage of GFP and OPH on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered E. coli can be applied in the form of a whole-cell biocatalyst and can be tracked by fluorescence during bioremediation. This strategy of codisplay should open a new dimension for the display of multiple functional moieties on the surface of a bacterial cell. Furthermore, a coculture comprised of the engineered E. coli and a natural p-nitrophenol (PNP) degrader, Ochrobactrum sp. strain LL-1, was assembled for complete mineralization of organophosphates (OPs) with a PNP substitution. The coculture degraded OPs as well as PNP rapidly. Therefore, the coculture with autofluorescent and mineralizing activities can potentially be applied for bioremediation of OP-contaminated sites.     

mRNA secondary structure modulates the translation of organophosphate hydrolase (OPH) in <Emphasis Type="Italic">E. coli</Emphasis>

Organophosphate hydrolases (OPHs), involved in hydrolytic cleavage of structurally diverse organophosphates are coded by a plasmid borne, highly conserved organophosphate degrading (opd) gene. An inverted repeat sequence found in the signal coding region of the opd gene was found to be responsible for inducing a stable stem loop structure with a ΔG of −23.1 kcal/mol. This stem loop structure has shown significant influence on the expression levels of organophosphate hydrolase (OPH) in E. coli. When the signal coding region comprising the inverted repeat sequence was deleted a ∼3.28 fold increase in the expression levels of OPH was noticed in E. coli BL21 cells. Mutations in the inverted repeat region, especially at the third position of the codon, to a non-complementary base destabilized the secondary structure of opd mRNA. When such opd variant, opd′ was expressed, the expression levels were found to be similar to expression levels coded by the construct generated by deleting the signal peptide coding region. Deletion of signal peptide did not influence the folding and activity of OPH. Though high level induction has resulted in accumulation of OPH as inclusion bodies, modulation of expression levels by reducing the copy number of the expression plasmid, inducer concentration and growth temperature has produced majority of the protein in soluble and active form.     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.     

Pathogenicity,Phylogenetic relationship and NGS based identification and assembly of tumorigenic Agrobacterium radiabacter plasmidic and chromosomic reads isolated from Prunus duclcis

Although many Agrobacterium radiobacter strains have already been identified, only a few genomes of strains belonging to genomovar G4 have been sequenced so far. In this study, we report the first virulent genome sequence of Agrobacterium radiobacter strain tun 183, which is highly virulent to almond specie. The genome size was estimated to be 5.53 Mb, with 57.9%GC content. In total, 6486 genes encoding proteins and 61 genes encoding RNAs were identified in this genome. Comparisons with the available sequenced genomes of genomovar G4 as well as with other A. sp. were conducted, revealing a hexapartite genome containing circular and linear chromosomes in addition to two accessory plasmids and a tumor inducing plasmid (pTi) in strain tun 183. The phylogenetic analysis of recA gene clearly showed the clustering of tun 183 strain within genomovar G4, supporting the monophyly within this genomovar.     

The structure of an enzyme–product complex reveals the critical role of a terminal hydroxide nucleophile in the bacterial phosphotriesterase mechanism

A detailed understanding of the catalytic mechanism of enzymes is an important step toward improving their activity for use in biotechnology. In this paper, crystal soaking experiments and X-ray crystallography were used to analyse the mechanism of the Agrobacterium radiobacter phosphotriesterase, OpdA, an enzyme capable of detoxifying a broad range of organophosphate pesticides. The structures of OpdA complexed with ethylene glycol and the product of dimethoate hydrolysis, dimethyl thiophosphate, provide new details of the catalytic mechanism. These structures suggest that the attacking nucleophile is a terminally bound hydroxide, consistent with the catalytic mechanism of other binuclear metallophosphoesterases. In addition, a crystal structure with the potential substrate trimethyl phosphate bound non-productively demonstrates the importance of the active site cavity in orienting the substrate into an approximation of the transition state.     

Heterologous Expression,Purification and Characterization of an Oligopeptidase A from the Pathogen <Emphasis Type="Italic">Leptospira interrogans</Emphasis>

Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn²⁺ at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.     

Expression of organophosphate hydrolase in the filamentous fungus Gliocladium virens

The broad-spectrum organophosphate hydrolase (OPH; EC 3.1.8.1) encoded by the organophosphate-degrading gene (opd) from Pseudomonas diminuta MG and Flavobacterium sp. ATCC 27551 possesses capabilities of both P-O bond hydrolysis (e.g. paraoxon) and P-F bond hydrolysis [e.g. sarin and diisopropylfluorophosphate (DFP)]. In the present study a 9.4-kb plasmid, pCL1, was used to transform the saprophytic fungus Gliocladium virens. pCL1 was derived from pJS294 by placing the fungal promoter (prom1) from Cochliobolus heterostrophus upstream and the trpC terminator from Aspergillus nidulans downstream of the opd gene. Southern analysis of restricted genomic DNA from various transformants indicated that integration occurred non-specifically at multiple sites. Western blot analysis of mycelial extracts from transformants confirmed the production of a processed form of the enzyme in the fungus. Maximal levels of OPH activity (rate of p-nitrophenol production from paraoxon) were observed after 168 h of culture and activity levels correlated with biomass production in mature vegetative growth.     

Protein localization in E. coli: Is there a common step in the secretion of periplasmic and outer-membrane proteins?

An E. coli strain carrying a fusion of the malE and lacZ genes is induced for the synthesis of a hybrid protein, consisting of the N-terminal part of the maltose-binding protein and the enzymatically active C-terminal part of β-galactosidase, by addition of maltose to cells. The secretion of the protein is initiated by the signal peptide attached to the N terminus of the maltose-binding protein sequence, but is not completed, presumably because the β-galactosidase moiety of the hybrid protein interferes with the passage of the polypeptide through the cytoplasmic membrane. Thus the protein becomes stuck to the cytoplasmic membrane. Under such conditions, periplasmic proteins, including maltose-binding protein (encoded by the malE gene) and alkaline phosphatase, and the major outer-membrane proteins, including OmpF, OmpA and probably lipoprotein, are synthesized as precursor forms with unprocessed signal sequences. This effect is observed within 15 min after high levels of induction are achieved. The simplest explanation for these results and those of pulse-chase experiments is that specific sites in the cytoplasmic membrane become progressively occupied by the hybrid protein, resulting in an inhibition of normal localization and processing of periplasmic and outer-membrane proteins. These results suggest that most of the periplasmic and outer-membrane proteins share a common step in localization before the polypeptide becomes accessible to the processing enzyme. If this interpretation is correct, we can estimate that an E. coli cell has roughly 2 × 10⁴ such sites in the cytoplasmic membrane. A system is described for detecting the precursor of any exported protein.     

Transposon-Like Organization of the Plasmid-Borne Organophosphate Degradation (opd) Gene Cluster Found in Flavobacterium sp.

Several bacterial strains that can use organophosphate pesticides as a source of carbon have been isolated from soil samples collected from diverse geographical regions. All these organisms synthesize an enzyme called parathion hydrolase, and in each case the enzyme is encoded by a gene (opd) located on a large indigenous plasmid. These plasmids show considerable genetic diversity, but the region containing the opd gene is highly conserved. Two opd plasmids, pPDL2 from Flavobacterium sp. and pCMS1 from Pseudomonas diminuta, are well characterized, and in each of them a region of about 5.1 kb containing the opd gene shows an identical restriction pattern. We now report the complete sequence of the conserved region of plasmid pPDL2. The opd gene is flanked upstream by an insertion sequence, ISFlsp1, that is a member of the IS21 family, and downstream by a Tn3-like element encoding a transposase and a resolvase. Adjacent to opd but transcribed in the opposite direction is an open reading frame (orf243) with the potential to encode an aromatic hydrolase somewhat similar to Pseudomonas putida TodF. We have shown that orf243 encodes a polypeptide of 27 kDa, which plays a role in the degradation of p-nitrophenol and is likely to act in concert with opd in the degradation of parathion. The linkage of opd and orf243, the organization of the genes flanking opd, and the wide geographical distribution of these genes suggest that this DNA sequence may constitute a complex catabolic transposon.     

Phosphate-bound structure of an organophosphate-degrading enzyme from Agrobacterium radiobacter

OpdA is a binuclear metalloenzyme that can hydrolyze organophosphate pesticides and nerve agents. In this study the crystal structure of the complex between OpdA and phosphate has been determined to 2.20 Å resolution. The structure shows the phosphate bound in a tripodal mode to the metal ions whereby two of the oxygen atoms of PO₄ are terminally bound to each metal ion and a third oxygen bridges the two metal ions, thus displacing the μOH in the active site. In silico modelling demonstrates that the phosphate moiety of a reaction product, e.g. diethyl phosphate, may bind in the same orientation, positioning the diethyl groups neatly into the substrate binding pocket close to the metal center. Thus, similar to the binuclear metallohydrolases urease and purple acid phosphatase the tripodal arrangement of PO₄ is interpreted in terms of a role of the μOH as a reaction nucleophile.     

Biochemical characterization and high-level production of oxidized polyvinyl alcohol hydrolase from Sphingopyxis sp. 113P3 expressed in methylotrophic Pichia pastoris

The Sphingopyxis sp. 113P3 gene oph, encoding oxidized polyvinyl alcohol hydrolase (OPH), was optimized with the preferred codons of Pichia pastoris and ligated into the pPIC9K vector behind the α-factor signal sequence. The vector was then transfected into P. pastoris GS115 and genomic integration was confirmed. Large-scale production of recombinant protein was performed by induction with 14.4 g/L methanol at 22 °C in a 3-L bioreactor. The maximal OPH activity obtained was 68.4 U/mL, which is the highest activity reported. The optimal pH and temperature of recombinant OPH were 8.0 and 45 °C, respectively. OPH activity was stable over a pH range of 5.0–8.5, and at a maximal temperature of 45 °C. The K _cat /K _m of recombinant OPH was 598 mM^?1 s^?1, which was 4.27-fold higher than that of recombinant OPH derived from Escherichia coli. The improved catalytic efficiency of OPH expressed in recombinant P. pastoris makes it favorable for industrial applications.     

A fluorogenic substrate for detection of organophosphatase activity

A new fluorogenic substrate for the specific detection of organophosphatase (OPase) activity has been designed and evaluated. Our results indicate that 7-diethylphospho-6,8-difluor-4-methylumbelliferyl (DEPFMU) is hydrolyzed specifically by the OPases, mammalian serum paraoxonase and bacterial organophosphorus hydrolase (OPH). The apparent K(m) of DEPFMU is 29 microM for OPH and 91 and 200 microM for the PON1 L(55)R(192) and PON1 L(55)Q(192) isoforms of human paraoxonase, respectively. DEPFMU-based assay systems are 10-100 times more sensitive for OPH and mammalian paraoxonase detection than existing methods. Importantly, DEPFMU is poorly hydrolyzed by both serum and cellular phosphatases and, therefore, may be used as part of a robust and sensitive assay for detecting not only purified, but also highly impure, preparations of OPase such as blood samples. The superior sensitivity of DEPFMU makes it potentially useful in the search for new enzymes that may hydrolyze nerve poisons such as sarin, soman, and VX, monitoring the decontamination of organophosphates (OPs) by OPH and determining serum paraoxonase activity which appears to be important for protection against atherosclerosis, sepsis, and OP toxicity.