Nadiya Bakhiya

Bernhard Monien

Heinz Frank

Albrecht Seidel

Prof. Dr Hansruedi R Glatt
email: glatt@dife.de

Renal organic anion transporters OAT1 and OAT3 mediate the cellular accumulation of 5-sulfooxymethylfurfural, a reactive, nephrotoxic metabolite of the Maillard product 5-hydroxymethylfurfural

Keywords: 5-hydroxymethylfurfural, 5-sulfooxymethylfurfural, nephrotoxicity, organic anion transporters

5-Hydroxymethylfurfural (HMF) is formed when sugars are acidified or heated. It is present at high levels in numerous foods. HMF is inactive in standard genotoxicity tests, but can be metabolized to a chemically reactive intermediate, 5-sulphooxymethylfurfural (SMF), which is mutagenic and carcinogenic. We recently found that direct parental administration of SMF to mice leads to abundant acute necrosis and proteinaceous casts in the proximal tubules as the dominating toxicological effect. Since proximal tubule cells actively mediate the excretion of many organic anions, we hypothesized that transporter-mediated uptake of SMF into the cells could be the reason for this selective organotoxicity. To test this hypothesis, we used human embryonic kidney (HEK293) cells stably expressing human (h) OAT1 or OAT3. SMF was a competitive inhibitor of p-aminohippurate uptake by hOAT1 and estrone sulfate uptake by hOAT3 with K i values of 225 µM and 1.5 mM, respectively. Moreover, the initial rates of SMF uptake were 5.2-and 3.1-fold higher in cells expressing hOAT1 and hOAT3, respectively, than in control HEK293 cells. Likewise, the sensitivity of hOAT1-and hOAT3expressing cells to SMF cytotoxicity was significantly higher than that of control cells, and was reduced by addition of probenecid, an inhibitor of OATs. Taken together, these results indicate that OAT1 and OAT3 mediate the uptake of SMF into proximal tubule cells and thereby may be involved in SMF-induced nephrotoxicity.

A c c e p t e d M a n u s c r i p t -4 -

Introduction

5-(Hydroxymethyl)-2-furfural (HMF) is one of the numerous compounds resulting from the heating of foods. It is formed when reducing hexose moieties are heated in the presence of amino acids or proteins (Maillard reaction) [START_REF] Mauron | The Maillard reaction in food; a critical review from the nutritional standpoint[END_REF] and via direct thermal dehydration of fructose, sucrose and glucose [START_REF] Antal | Mechanism of formation of 5-(hydroxymethyl)-2-furaldehyde from D-fructose and sucrose[END_REF]. HMF is present at high levels in many foods, with particularly high concentrations found in caramel products (up to 9.5 g/kg), dried fruit (especially plums), old Port and Madeira wines, and balsamic vinegar [START_REF] Bachmann | 5-Hydroxymethyl-2-furfural (HMF) in Lebensmitteln[END_REF][START_REF] Murkovic | Analysis of 5-hydroxymethylfurfual in coffee, dried fruits and urine[END_REF][START_REF] Husøy | Dietary exposure to 5-hydroxymethylfurfural from Norwegian food and correlations with urine metabolites of short-term exposure[END_REF]. However, coffee may be a major source of HMF intake in many subjects due to the high amount consumed [START_REF] Husøy | Dietary exposure to 5-hydroxymethylfurfural from Norwegian food and correlations with urine metabolites of short-term exposure[END_REF]. The estimated daily intake is in the range of 30-150 mg per person [START_REF] Ulbricht | A review of 5-hydroxymethylfurfural (HMF) in parenteral solutions[END_REF][START_REF] Janzowski | 5-Hydroxymethylfurfural: assessment of mutagenicity, DNA-damaging potential and reactivity towards cellular glutathione[END_REF]. Although HMF is inactive in standard in vitro genotoxicity tests [START_REF] Janzowski | 5-Hydroxymethylfurfural: assessment of mutagenicity, DNA-damaging potential and reactivity towards cellular glutathione[END_REF][START_REF] Glatt | Health risks by 5-hydroxymethylfurfural (HMF) and related compounds[END_REF], it induced and promoted preneoplastic lesions, aberrant crypt foci, in rat colon [START_REF] Zhang | Initiation and promotion of colonic aberrant crypt foci in rats by 5-hydroxymethyl-2-furaldehyde in thermolyzed sucrose[END_REF] and initiated papillomas in mouse skin [START_REF] Surh | 5-Sulfooxymethylfurfural as a possible ultimate mutagenic and carcinogenic metabolite of the Maillard reaction product, 5hydroxymethylfurfural[END_REF]. Furthermore, in a long-term carcinogenicity study conducted by National Toxicology Program, HMF was found to increase the incidence of hepatocellular adenomas in female mice [START_REF]Toxicology and carcinogenesis studies of 5-(hydroxymethyl)-2-furfural[END_REF].

Numerous chemicals are mutagenic or carcinogenic after metabolic activation to chemically reactive intermediates. HMF is converted to a chemically reactive allylic ester, 5sulfooxymethylfurfural (SMF, structural formula depicted in Fig. 1) by sulfotransferases (SULT), enzymes ignored in standard in vitro genotoxicity tests. HMF showed mutagenic activity when tested in bacterial and mammalian target cells engineered for expression of human SULT1A1 [START_REF] Glatt | Health risks by 5-hydroxymethylfurfural (HMF) and related compounds[END_REF]. SMF, unlike HMF, formed DNA adducts in cell-free systems and was mutagenic to bacterial and mammalian cells without requiring an activating system [START_REF] Glatt | Health risks by 5-hydroxymethylfurfural (HMF) and related compounds[END_REF][START_REF] Surh | Activation of the Maillard reaction product 5-(hydroxymethyl)furfural to strong mutagens via allylic sulfonation and chlorination[END_REF].

When administered topically to mouse skin, SMF initiated papillomas with higher activity than HMF [START_REF] Surh | 5-Sulfooxymethylfurfural as a possible ultimate mutagenic and carcinogenic metabolite of the Maillard reaction product, 5hydroxymethylfurfural[END_REF]. In a recent study conducted in our laboratory SMF was found to be strongly nephrotoxic in the mouse in vivo (F. Taugner, S. Florian and H. R. Glatt, manuscript in preparation). Male FVB/N mice which received a single dose of SMF (250 mg SMF /kg body mass, i.p.) died or were moribund 5-11 days after the treatment. Histopathological analyses revealed that SMF induced moderate damage in liver and most impressive damage in the kidneys, particularly in the proximal tubules, where abundant necrosis and proteinaceous casts were observed. While nearly all proximal tubules in SMF-exposed animals were destroyed, distal tubules and glomeruli were less affected. The molecular mechanism underlying this selective toxicity of SMF in proximal tubules is unknown.

An important physiological function of the proximal tubule is the secretion of a large variety of organic anions, including endogenous catabolites, drugs and other xenobiotics [START_REF] Russel | Molecular aspects of renal anionic drug transport[END_REF]. The organic anion transporters OAT1 and OAT3 are localized at the basolateral membrane of proximal tubule cells and mediate the concentrative uptake of their substrates from blood into renal proximal tubule cells, which is the rate-limiting step in this excretion system [START_REF] Burckhardt | Transport of organic anions across the basolateral membrane of proximal tubule cells[END_REF]. Genes of both transporters were cloned from several species, including rat and man, and are highly conserved across species [START_REF] Burckhardt | Transport of organic anions across the basolateral membrane of proximal tubule cells[END_REF][START_REF] Robertson | Human renal organic anion transporters: characteristics and contributions to drug and drug metabolite excretion[END_REF]. Both transporters show overlapping substrate specificities and share the driving force -exchange of organic anions against intracellular α-ketoglutarate.

Moreover, the activity of OAT1 and OAT3 has been associated with proximal tubular injury due to the accumulation of toxicants, such as uremic toxins, mercuric species and ochratoxin A [START_REF] Sweet | Organic anion transporter (Slc22a) family members as mediators of toxicity[END_REF], as well as genotoxic benzylic sulfates formed from alkylated polycyclic aromatic hydrocarbons [START_REF] Bakhiya | Uptake of chemically reactive, DNA-damaging sulfuric acid esters into renal cells by human organic anion transporters[END_REF].

The aim of the present investigation was to determine the possible involvement of OAT1 and OAT3 in the uptake, accumulation and toxicity of SMF in renal cells. To this end, we used A c c e p t e d M a n u s c r i p t -6 -human embryonic kidney cells (HEK293) stably expressing human (h) OAT1 or OAT3 to assess both uptake and toxicity of SMF.

Materials and methods

Chemicals

5-Hydroxymethylfurfural, sulfur trioxide-pyridine complex, sodium methoxide, and the anhydrous solvents tetrahydrofuran, diethyl ether, and dimethylformamide were purchased from Sigma-Aldrich (Taufkirchen, Germany) and used as received. All solvents for UPLC were purchased from Carl Roth GmbH (Karlsruhe, Germany). Cell culture reagents were from Gibco Invitrogen (Karlsruhe, Germany), unless specified otherwise. [6,7-3 NMR spectra were obtained on a Bruker AV400 spectrometer at the University of Hamburg.

Melting points (mp) were determined on a Büchi 510 apparatus.

Synthesis of SMF

5-Hydroxymethylfurfural (0.032 g; 0.25 mmol) was dissolved in 1 ml anhydrous tetrahydrofuran and this solution was cooled to 0°C. After addition of a solution of 80 mg sulfur trioxide-pyridine complex (0.5 mmol) in 0.65 ml anhydrous N,N-dimethylformamide the mixture was stirred at 0°C for 4 hours. Then a solution of 40 mg (0.74 mmol) sodium The purity of SMF was greater than 99 % as determined by liquid chromatography-mass spectrometry (see section 2.5).

Tissue culture

hOAT1 and hOAT3 were stably expressed in the human embryonic kidney cell line HEK293 as described previously [START_REF] Bakhiya | Uptake of chemically reactive, DNA-damaging sulfuric acid esters into renal cells by human organic anion transporters[END_REF]. Cells were grown in flasks containing Dulbecco's modified minimum essential medium (high glucose) supplemented with fetal bovine serum (10 %, Biochrom AG, Berlin, Germany), penicillin (100 units/ml) and streptomycin (100 µg/ml). The medium for the recombinant cells, but not for parental HEK293 cells, additionally contained hygromycin (175 µg/ml). Cultures were maintained in a humidified atmosphere containing 5 % CO 2 at 37°C. Cultures were split in a 1:5 ratio every third to forth day.

Inhibition of uptake of standard substrates

Cells were seeded in 24-well plates (2 x 10 5 cells in 1 ml medium per well) two days before the experiment. Uptake of [ 3 H]p-aminohippurate (for OAT1) and [ 3 H]estrone sulfate (for hOAT3) was assayed at 37°C in Ringer's solution (130 mM NaCl, 4 mM KCl, 1 mM CaCl 2 , 1 mM MgSO 4 , 20 mM HEPES, 1 mM NaH 2 PO 4 , 18 mM glucose, pH 7.4) for 2 min in the absence or presence of SMF. The uptake was terminated by three washes with 0.5 ml of icecold Ringer's solution. Cells were then solubilized in 0.5 ml of 1 N NaOH. After A c c e p t e d M a n u s c r i p t -8 -neutralization with 0.5 ml of 1 N HCl, their 3 H content was assayed by liquid scintillation counting. The results were standardized to the level of protein determined using the bicinchoninic acid assay (Pierce, Rockford, USA) with bovine serum albumin as the standard.

For quantifying the inhibitory activity, we used varying concentrations of the substrate (10 and 50 µM [ 3 H]p-aminohippurate for hOAT1; 50 and 250 nM [ 3 H]estrone sulfate for hOAT3) and SMF (≥ 5 concentrations, up to 1 mM for hOAT1 and 2 mM for hOAT3). Data were plotted and analyzed according to Dixon [START_REF] Dixon | The determination of enzyme inhibitor constants[END_REF].

LC-MS/MS analysis of SMF uptake into cells

Cells were incubated in Ringer's solution containing 250 µM SMF at room temperature for 1-15 min. After three washes with ice-cold Ringer's solution, the cells were lysed with 0.25 ml 1 N NaOH and, after neutralization with 0.25 ml of 1 N HCl and protein precipitation with 1 ml of ice-cold 2-propanol, mixed thoroughly and centrifuged at 15,000 g for 15 min. The clear supernatant was analyzed using an Acquity ultra performance liquid chromatography (UPLC) System (Waters, Milford, Massachussetts, USA) with a UPLC BEH Phenyl column (1.7 µm, 2.1 × 100 mm, Waters). Samples of 4 µl were injected, and SMF was eluted with 10 mM ammonium acetate/methanol (95:5) at 0.35 ml/min flow rate. The UPLC was connected to a Quattro Premier XE tandem quadrupole mass spectrometer (Waters Micromass, Manchester, UK) with an electrospray interface operated in the negative ion mode. Two fragmentation reactions of SMF were monitored, yielding the sulfate ion radical (204.9 → 96) and the protonated sulfonate ion (204.9 → 81). The tune parameter were as follows: temperature of the electrospray source 120°C; desolvation temperature 500°C; desolvation gas: nitrogen (850 l/h); cone gas: nitrogen (100 l/h); collision gas: argon (indicated cell pressure ~ 5•10 -3 mbar). For the fragmentation of SMF, collision energies were 18 and 22 eV A c c e p t e d M a n u s c r i p t -9 -for the transitions 204.9 → 81 and 204.9 → 96, respectively. The dwell time was set to 100 ms, and capillary voltage was set to 0.35 kV. The cone and RF1 lens voltages were 32 V and 0.1 V, respectively. SMF was quantified using an external calibration line prepared in water/2-propanol (1:3). The calibration curves were linear in the range between 500 nM (2 pmol/injection) and 1 nM (4 fmol/injection) SMF (r 2 ≥ 0.993). Details of the method has been published elsewhere [START_REF] Monien | Conversion of the common food constituent, 5-hydroxymethylfurfural, into a mutagenic and carcinogenic sulfuric acid ester in the mouse in vivo[END_REF].

[ 3 H]Thymidine-incorporation proliferation assay

Cells (1 x 10 5 cells per well) were seeded in 24-well plate 24 h before starting exposure. Then, they were incubated at 37°C for 1 h in the absence or presence of various concentrations of SMF (0.1-1.6 mM). After the incubation, cells were washed twice with medium. [ 3 H]Thymidine (0.2 µCi/ml in culture medium) was incorporated at 37°C for 1 h. The cells were fixed with 5 % trichloroacetic acid (30 min at 4°C), washed twice with icecold PBS and once with of ice-cold 95 % ethanol to remove unused [ 3 H]thymidine. For the solubilization of the genomic DNA, the cells were incubated with 0.5 ml of 1 N NaOH overnight. After neutralization with 0.5 ml of 1 N HCl, the 3 H content was assayed by liquid scintillation counting.

Results

Inhibition of hOAT1 and hOAT3 by SMF

SMF was tested for its ability to inhibit hOAT-mediated uptake of model substrates, paminohippurate for hOAT1 and estrone sulfate for hOAT3 in recombinant embryonic kidney A c c e p t e d M a n u s c r i p t -10 -cells. As shown in Fig. 2, SMF inhibited substrate uptake by both hOATs. The effect increased with the concentration of SMF. The sensitivities of hOAT1-and hOAT3-mediated substrate uptakes to SMF inhibition were different. At a concentrations of 0.5 and 1 mM, SMF inhibited p-aminohippurate (10 µM) uptake by hOAT1-expressing cells by 68 % and 79 %, respectively (Fig. 2A). In the presence of the same SMF concentrations (0.5 and 1 mM) estrone sulfate (50 nM) uptake by hOAT3-cells was inhibited somewhat less (by 27 % and 45 %, respectively, Fig. 2B).

hOAT1-and hOAT3-mediated uptake of SMF into cells

To test whether SMF is transported by hOAT, we exposed parental HEK293 and transporterexpressing cells to SMF (250 µM) for 1-15 min and then analyzed the intracellular SMF content by UPLC-MS/MS. The analytical method used was highly specific and sensitive (limit of detection ~ 5 fmol per injection). As shown in Fig. 4A, the uptake was linear with time for at least 4-10 min in all three cell lines. The initial slopes amounted to 1.3, 6.6 and 3.9 nmol/mg protein/min for control, hOAT1-and hOAT3-expressing cells respectively. Thus, expression of hOAT1 and hOAT3 enhanced the uptake rates by a factor of 5.2 and 3.1,

A c c e p t e d M a n u s c r i p t

-11 -respectively. Moreover, the SMF uptake by hOAT-expressing cells was inhibited by addition of probenecid (a known inhibitor of OAT) to the incubation medium, indicating that this uptake was hOAT-mediated (Fig. 4B).

Influence of hOAT1 and hOAT3 expression on cellular toxicity of SMF

As shown in Fig. 5, hOAT1 and hOAT3-expressing were markedly more sensitive than control cells to toxic effects of SMF. The difference was significant for both transporters starting from 0.2 mM SMF concentration. The enhanced cytotoxicity of hOAT-expressing cells to SMF was directly related to the activity of the expressed transporters, as demonstrated using probenecid, an inhibitor of OATs. Probenecid markedly reduced of the cytotoxicity of SMF (0.4 mM) in the hOAT-expressing cells (Fig. 6). Survival of hOAT1-expressing cells was elevated by probenecid from 49 % to 71 %. Similarly, survival of hOAT3-expressing cells was increased from 55 % in the absence to 74 % in the presence of probenecid.

Discussion

HMF can be activated to a chemically reactive and genotoxic metabolite, SMF. In a recent in vivo study we detected strong toxicity of SMF to renal proximal tubule cells leading to death in most animals after a single dose of 250 mg/kg. At the next lower dose (125 mg/kg), animals developed an atypical renal hyperplasia. The proximal tubule cells are the primary sites of OAT1 and OAT3 expression, and previous studies suggest that these transporters could participate in accumulation of reactive sulfo conjugates in this tissue.

A c c e p t e d M a n u s c r i p t -12 -

The data reported here provide molecular evidence that hOAT1 and hOAT3 can mediate the uptake of SMF into renal cells. Both uptake and toxicity of SMF were significantly higher in HEK293 cells expressing hOAT1 or hOAT3 than in control cells lacking these transporters. Furthermore, both uptake and toxicity were reduced by the OAT inhibitor probenecid, indicating that SMF is a substrate of hOAT1 and hOAT3. Therefore, OAT1 and OAT3 seem to provide specific paths for the entry of SMF into renal cells, leading to proximal tubule damage. The affinities of SMF interaction with hOAT1 and hOAT3, measured in the inhibition study, were considerably different. The K i value of hOAT1 for SMF was in the range of 225 µM, while the affinity of the hOAT3 to SMF was approximately 7 times lower (K i value ~ 1.5 mM), suggesting that this transporter contributes less to cellular SMF uptake than hOAT1.

As shown in rodents, HMF is well absorbed from the gastro-intestinal tract, rapidly metabolized, and excreted primarily in urine. The principal urinary metabolites are 5hydroxymethyl-2-furoic acid and its glycine conjugate and furan-2,5-dicarboxylic acid [START_REF] Jellum | The presence of furan derivatives in patients receiving fructose-containing solutions intravenously[END_REF][START_REF] Germond | Rapid and complete urinary elimination of [ 14 C]-5-hydroxymethyl-2-furaldehyde administered orally or intravenously to rats[END_REF][START_REF] Godfrey | Distribution and metabolism of (5-hydroxymethyl)furfural in male F344 rats and B6C3F1 mice after oral administration[END_REF]. SMF was not detected in urine. However, SMF is chemically reactive, making its detection in biological samples difficult. The half-life time of SMF in aqueous solutions at 37°C amounts to nearly 2 h. Recent studies conducted in our laboratory demonstrate the formation of SMF from HMF in the mouse in vivo [START_REF] Monien | Conversion of the common food constituent, 5-hydroxymethylfurfural, into a mutagenic and carcinogenic sulfuric acid ester in the mouse in vivo[END_REF]. SMF was detected in blood samples of FVB/N mice after intravenous administration of HMF. From a dose of 100 mg HMF/kg body mass, approximately 0.04 % HMF was metabolized to SMF and exported into the circulation; additional SMF may have been formed and reacted with cellular structures or hydrolyzed back to HMF at its side of formation not reaching the circulation. Several human SULT forms catalyze the sulfo conjugation of HMF with higher catalytic efficiency than murine enzymes; this is true in particular for SULT1A1, the most abundant SULT form in the A c c e p t e d M a n u s c r i p t -13 -human organism [START_REF] Glatt | Health risks by 5-hydroxymethylfurfural (HMF) and related compounds[END_REF]. Therefore, it may be speculated that a higher percentage of HMF is converted to SMF in humans than in mice. Taking into consideration that the daily intake of HMF exceeds that of other food-borne pro-genotoxicants, such as acrylamide or heterocyclic amines, by factors of 10 3 and 10 5 , respectively, conversion of a minute fraction to a reactive metabolite still could be toxicologically relevant.

Recently HMF was orally administrated to B6C3F1 mice and F344/N rats to study its subacute, subchronic and chronic toxicity [START_REF]Toxicology and carcinogenesis studies of 5-(hydroxymethyl)-2-furfural[END_REF]. In a 3-month study, the incidence of cytoplasmic alterations of the kidney was significantly increased in male mice receiving 188 or 375 mg/kg HMF. In a 2-year carcinogenicity study, HMF showed evidence of carcinogenic activity, clearly increasing the incidence of hepatocellular adenomas in female mice receiving 188 or 375 mg/kg HMF. Thus, SMF formed in vivo in mice seems to cause the main damage at the site of its formation, the liver; probably only a minor part of SMF is exported to the blood. The situation in humans could be different, taking into consideration the speciesdependent differences in tissue distribution of HMF-activating SULTs, which are focused to the liver in mice, but widely expressed in numerous tissues in humans [START_REF] Glatt | ditor. Handbook of Enzyme Systems that Metabolise Drugs and Other Xenobiotics[END_REF][START_REF] Teubner | Identification and localization of soluble sulfotransferases in the human gastrointestinal tract[END_REF]. Furfuryl alcohol is another common Maillard product. It only differs from HMF by the absence of the aldehyde group in 2 position. Like HMF, furfuryl alcohol is also activated to a mutagenic sulfo conjugate, furfuryl sulfate, by human SULTs [START_REF] Glatt | Health risks by 5-hydroxymethylfurfural (HMF) and related compounds[END_REF]. Two-year carcinogenicity studies in male and female Fischer 344/N rats and B6C3F 1 mice had been conducted with furfuryl alcohol administrated by inhalation [START_REF] Ntp | Toxicology and carcinogenesis studies of furfuryl alcohol (CAS No.98-00-0) in F344/N rats and B6C3F 1 mice (inhalation studies)[END_REF]. Notably, the incidence of renal tubule neoplasms was increased in male mice exposed to furfuryl alcohol, and the severity of nephropathy (typical for old mice and rats) was enhanced in both sexes of the rat as well as in male mice. We therefore suspect that the renal toxicity and carcinogenicity of furfuryl alcohol A c c e p t e d M a n u s c r i p t -14 -was mediated by its metabolite furfuryl sulfate, which is structurally related to SMF and thus could also be accumulated in the renal tubule cells by organic anion transporters.

Findings with some other chemicals indicate that kidneys might be a relatively common target tissue of reactive sulfo conjugates. 1-Sulfooxymethylpyrene is an ultimate mutagen and carcinogen formed from an abundant polycyclic aromatic hydrocarbon, 1-methylpyrene. After treating rats with 1-sulfooxymethylpyrene or its metabolic precursor, 1-hydroxymethylpyrene, the highest level of DNA adducts was detected in kidney [START_REF] Glatt | Metabolic formation, distribution and toxicological effects of reactive sulphuric acid esters[END_REF]. Further in vitro studies showed that 1-sulfooxymethylpyrene is an excellent substrate for OAT1 and OAT3 [START_REF] Bakhiya | Uptake of chemically reactive, DNA-damaging sulfuric acid esters into renal cells by human organic anion transporters[END_REF].

Renal cell carcinomas account for approximately 3 % of all adult malignancies [START_REF] Martel | Renal cell carcinoma: current status and future directions[END_REF]. The clear cell carcinoma, arising from proximal tubular cells, makes up 70 to 80 % of renal neoplasias.

The incidence of renal cell carcinomas varies more than 10-fold between different countries and has been increasing in various countries, suggesting an involvement of exogenous factors.

It would be interesting to know whether, and to which extent, HMF, furfuryl alcohol, alkylated polycyclic aromatic hydrocarbons and other SULT-dependent promutagens contribute to the induction of clear cell carcinomas.

In conclusion, in the present study we provide evidence for the involvement of organic anion transporters (OATs) in the renal accumulation of SMF. These transport characteristics could be responsible for the selective damage of renal proximal tubules by this reactive metabolite.

  Abbreviations: HMF, 5-hydroxymethylfurfural; hOAT, human organic anion transporter;

  35 ml methanol was added and the resulting mixture was centrifuged. The supernatant was added dropwise to 80 ml of anhydrous diethyl ether. The formed precipitate was filtered off, washed with anhydrous diethyl ether, and dried under vacuum to give a yield of 0.043 g SMF (75% yield); mp 128 °C (dec); 1 H-NMR (400 MHz, DMSO-d6) δ [ppm] 9.63 (s, 1, CHO), 7.54 (d, 1, H 3 , J 3,4 = 3.6 Hz), 6.75 (d, 1, H 4 , J 4,3 = 3.6 Hz), 4.83 (s, 2, -CH 2 -O).

  Subsequently, we studied the inhibition kinetics of SMF using different concentrations of the model substrates and SMF. The K i values were determined from Dixon plots. Representative plots are shown in Fig. 3. Similar results were obtained in repeat experiments. SMF competitively inhibited the organic anion transport mediated by both transporters. The mean K i value (± SE) of SMF for hOAT1 was determined at 225 ± 22 µM (245, 248 and 182 µM in three experiments). The corresponding value for hOAT3 was 1.46 ± 0.04 mM (1.38, 1.53 and 1.47 mM in three experiments).

Figure legends FIG. 1 .FIG. 2 .FIG. 3 .

 legends123 Figure legends

Fig. 4 .FIG. 5 .FIG. 6 .

 456 Fig. 4. Uptake of SMF by human OAT-expressing cells. (A) Control HEK293 cells and

  Figure_1

Figure_5A

  Figure_5

Figure_6

  Figure_6

Acknowledgements


Uttamkumar Samanta

Stephen D Kirby

Prabhavathi Srinivasan

Douglas M Cerasoli

Brian J Bahnson
email: bahnson@udel.edu.

Crystal Structures of Human Group-VIIA Phospholipase A2 Inhibited by Organophosphorus Nerve Agents Exhibit Non-aged Complexes

Keywords: phospholipase A2, Lp-PLA2, PAF-AH, organophosphate, nerve agent AChE, acetylcholinesterase, AH, acetylhydrolase, BChE, butyrylcholinesterase

Protein Data Bank entry codes 3F9C, 3F96, 3F97, and 3F98 are non-aged complexes of human group-VIIA phospholipase A2 with the pesticide diisopropylfluorophosphate or the nerve agents

The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun.

The sarin and soman complexes displayed a racemic mix of P R and P S stereoisomers at the Pchiral center. The tabun complex displayed only the P R stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme.

The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents. Introduction

The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2, EC 3.1.1.47) hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain [START_REF] Schaloske | The phospholipase A2 superfamily and its group numbering system[END_REF]. Originally the enzyme was identified by its ability to hydrolyze the acetyl group from the sn-2 position of the signaling molecule platelet activating factor (PAF), and was therefore referred to as PAF acetylhydrolase [START_REF] Tjoelker | Plasma platelet-activating factor acetylhydrolase is a secreted phospholipase A2 with a catalytic triad[END_REF]. In addition to its suspected role in reducing PAF levels, gVIIA-PLA2 hydrolyzes other pro-inflammatory agents such as oxidized lipids of LDL particles [START_REF] Stremler | Human plasma plateletactivating factor acetylhydrolase. Oxidatively fragmented phospholipids as substrates[END_REF][START_REF] Davis | Electrospray ionization mass spectrometry identifies substrates and products of lipoprotein-associated phospholipase A(2) in oxidized human low density lipoprotein[END_REF].

Physiologically, gVIIA-PLA2 is associated to both LDL and HDL particles, and is also referred to as lipoprotein associated PLA2 (Lp-PLA2) [START_REF] Macphee | An evolving story of lipoprotein-associated phospholipase A2 in atherosclerosis and cardiovascular risk prediction[END_REF]. The enzyme is tightly associated with lipoprotein particles, and is thought to access substrates from both the lipoprotein surface and from the aqueous phase [START_REF] Min | Membranebound plasma platelet activating factor acetylhydrolase acts on substrate in the aqueous phase[END_REF][START_REF] Min | Platelet-activating factor acetylhydrolases: broad substrate specificity and lipoprotein binding does not modulate the catalytic properties of the plasma enzyme[END_REF][START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF].

Organophosphorous (OP) compounds are believed to exhibit their toxicity primarily through the inhibition of acetylcholinesterase (AChE), which is responsible for degrading the neurotransmitter acetylcholine. Nerve agents such as sarin, soman, and tabun (Fig. 1) pose a risk to human health. Additionally, a larger super-family of serine hydrolases, including gVIIA-PLA2, have been shown to be inactivated by OPs, with potential clinical consequences [START_REF] Casida | Organophosphate toxicology: safety aspects of nonacetylcholinesterase secondary targets[END_REF]. As depicted in Fig. 1, the nucleophilic serine residue reacts with OPs by a S N 2 mechanism with a trigonal bipyramidal transition state, and stereoinversion occurs at the chiral phosphorus atom [START_REF] Millard | Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level[END_REF]. Depending on the enzyme, the initial complex can yield one of several results: reactivation through spontaneous hydrolysis, reactivation with a strong nucleophile, dealklyation of the OP adduct, or maintenance of a stable complex. When bound to AChE, OP compounds with a branched side-chain at the C α adjacent to the phosphoester typically undergo a rapid dealkylation process referred to as aging [START_REF] Michel | Ageing and dealkylation of Soman (pinacolylmethylphosphonofluoridate)-inactivated eel cholinesterase[END_REF][START_REF] Harris | Dealkylation and loss of capacity for reactivation of cholinesterase inhibited by sarin[END_REF]. The aging process in AChE is thought to be irreversible, as A c c e p t e d M a n u s c r i p t 6 the covalently inhibited enzyme cannot typically be reactivated by nucleophiles to regenerate enzymatic activity. Alternatively, the non-aged complex can undergo a reactivation by attack of an activated water molecule or an alternative activated nucleophile (e.g., oxime). Efforts have focused on engineering serine hydrolase enzymes to hydrolyze pesticides and nerve agents, and therefore function as catalytic OP bioscavengers [START_REF] Broomfield | Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization[END_REF]. OP nerve agents, such as sarin, soman, tabun or VX are particularly toxic to humans and are commonly referred to as chemical warfare agents. The gVIIA-PLA2 enzyme is an intriguing system to explore OP reactivity, as it is present in human blood, which is the path nerve agents take to their ultimate target of nerve synapse AChE, and the most suitable site for detoxifying OPs under the bioscavenger paradigm.

One approach to develop an OP bioscavenger is to use structures of serine hydrolases complexed with the nerve agents of interest as a starting point to engineer hydrolase activity.

Several systems have been examined, which include structural information of both aged and nonaged complexes. Crystal structures exist for non-human AChE complexed with sarin, VX and DFP in both aged and non-aged forms [START_REF] Millard | Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level[END_REF][START_REF] Hornberg | Crystal structures of acetylcholinesterase in complex with organophosphorus compounds suggest that the acyl pocket modulates the aging reaction by precluding the formation of the trigonal bipyramidal transition state[END_REF][START_REF] Millard | Reaction Products of Acetylcholinesterase and VX Reveal a Mobile Histidine in the Catalytic Triad[END_REF]. Additionally, the non-aged structure of human butyrylcholinesterase (BChE) was reported in complex with the OP echothiophate [START_REF] Nachon | Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging[END_REF] and tabun [START_REF] Carletti | Aging of Cholinesterases Phosphylated by Tabun Proceeds through O-Dealkylation[END_REF]. Although VX and echothiophate have a propensity to age, the leaving group is unbranched at the C α position, suggesting that this is a relatively slow reaction in both AChE and BChE. More recently, human carboxylesterase was reported to form non-aged complexes with the OPs soman and tabun [START_REF] Fleming | Crystal structures of human carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun[END_REF]. Additionally, bovine group-VIII (gVIII) PLA2 was reported by us to form non-aged complexes with sarin and soman [START_REF] Epstein | Crystal structures of brain group-VIII phospholipase A2 in nonaged complexes with the organophosphorus nerve agents soman and sarin[END_REF].

The gVIIA-PLA2 enzyme, which is structurally, functionally and physiologically unrelated [START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF] to the gVIII-PLA2 enzyme is bound to lipoproteins. It has great promise as a potential OP bioscavenger, as it is naturally present in human blood. We recently reported the A c c e p t e d M a n u s c r i p t 7 crystal structure of gVIIA-PLA2 in a ligand-free form and following reaction with the OP insecticide paraoxon [START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF]. In our present work, we report the structures of gVIIA-PLA2 following reaction with the OP compounds sarin, soman, tabun and DFP to a resolution of 2.1 Å, 1.7 Å, 1.7 Å, and 2.3 Å, respectively. Each structure reported provides convincing evidence of non-aged complexes, and in the case of the tabun complex a single stereoisomer in the crystal structure.

Furthermore, results reported from mass spectrometry experiments corroborate the crystallographic results of non-aged OP complexes, and show evidence for a lack of stereoselectivity of reaction of sarin with gVIIA-PLA2.

Materials and Methods

Preparation of protein

Crystallization experiments utilized human gVIIA-PLA2 (PAFase, residues 47-429, NCBI accession Q13093) which was overexpressed in E. coli and obtained from ICOS Corporation.

Details of the protein preparation of the ICOS-produced protein and initial crystallization of the ligand-free form of human gVIIA-PLA2 have been reported previously [START_REF] Samanta | Crystallization and preliminary x-ray crystallographic analysis of human plasma platelet activating factor acetylhydrolase[END_REF]. Mass spectrometry work utilized both PAFase from ICOS and human gVIIA-PLA2 protein expressed in our lab using the following methods. The full length clone of human gVIIA-PLA2 was purchased from Invitrogen (NCBI accession BC038452), the cDNA was amplified by PCR, and subcloned into the expression plasmid pGEX-4T3 (GE Healthcare) utilizing SalI (Invitrogen) and NotI (New England Biolabs) restriction enzyme sites. The recombinant gVIIA-PLA2 construct included residues 42-441 of human gVIIA-PLA2 with an N-terminal extension of 7 additional residues which remained after thrombin cleavage. This construct represents a full length construct of gVIIA-PLA2. The N-terminus start of gVIIA-PLA2 found in human blood is heterogeneous, A c c e p t e d M a n u s c r i p t 8 with a mixture of N-termini at positions Ser35, Ile42 or Lys55 [START_REF] Tjoelker | Plasma platelet-activating factor acetylhydrolase is a secreted phospholipase A2 with a catalytic triad[END_REF]. The natural C-terminus of gVIIA-PLA2 is residue Asn441. The first 7 amino acids of this construct (SPNSRVD) are nonnative sequence coming from the multiple cloning site of the PGEX4T-3 vector into which the gVIIA-PLA2 sequence was cloned. The GST-gVIIA-PLA2 fusion protein was over-expressed in E. coli strain BL21. Ten milliliters of the overnight culture was inoculated into 1 L of fresh LB media containing 75 µg/ml ampicillin (Sigma-Aldrich) and grown at 37 ˚C until an optical density of 0.5-0.6 was reached at 600 nm. Protein expression was induced by the addition of 0.5 mM IPTG (Sigma-Aldrich) at 37 ˚C for 4 hrs. Cells were harvested by centrifuging at 5,000 rpm for 10 min at 4 ˚C. Cells were lysed by sonication in 50 mM Tris (Fisher Sci.), pH 7.8, 100 mM NaCl (Fisher Sci.), 5 mM DTT (Sigma-Aldrich), 1 mM PMSF (Sigma-Aldrich), 0.1 mg/ml lysozyme (Sigma-Aldrich). Cell lysis was followed by detergent extraction with 0.2% (w/v)

Triton DF-16 (Sigma-Aldrich) for 30 min at 4 ˚C. The fusion protein was purified by affinity chromatography using glutathione sepharose (GE Healthcare). The GST-tag was cleaved from the fusion protein via on-column cleavage using thrombin protease (Enzyme Research Laboratories). Digestion was typically allowed to proceed overnight at 4 ˚C. After digestion, cleaved gVIIA-PLA2 was eluted with 50 mM Tris, pH 7.8, 500 mM NaCl, 1 mM EDTA (Sigma-Alrich), 1 mM DTT, 0.2% (w/v) Triton DF-16. Thrombin was removed from the purified protein by affinity chromatography using a benzamidine sepharose cartridge column (GE Healthcare).

Purified protein was concentrated to 3-4 mg/ml using centrifugal filter units (Millipore).

Concentrated protein was dialyzed against 50 mM Tris, pH 7.8, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2% (w/v) Triton DF-16 before storage at 4 ˚C.

Inhibition and crystallization of gVIIA-PLA2

A c c e p t e d M a n u s c r i p t 9 Samples of gVIIA-PLA2 (PAFase, residues 47-429) were prepared for crystallization as previously described [START_REF] Samanta | Crystallization and preliminary x-ray crystallographic analysis of human plasma platelet activating factor acetylhydrolase[END_REF] and concentrated to 4 mg/ml. Initially ligand-free protein crystals of gVIIA-PLA2 were grown by the methods described previously [START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF]. Then crystals were transfered into a reservoir solution containing 27 mM BOG, 1.0 mM DTT and a 4 mM DFP solution. Protein crystals were soaked in this DFP solution for 20 min, and then flash frozen into liquid nitrogen.

Crystal structures of OP complexes

X-ray diffraction data for tabun gVIIA-PLA2 crystals were collected at beamline 19ID, whereas gVIIA-PLA2 sarin and soman complex crystal data were collected at beamline 24ID of the Advanced Photon Source at the Argonne National Laboratory. The DFP-inhibited protein X-ray diffraction data were collected with a Rigaku RU-H3R anode generator and a RAXIS IV image plate area detector of our in-house system. The program HKL2000 [START_REF] Otwinowski | Processing of x-ray diffraction data collected in oscillation mode[END_REF] was used to process data in the space group C2 for complexes of sarin, soman and DFP, and the tabun inhibited complex data were processed in the space group of C222 1 . The structure of gVIIA-PLA2 (PDB entry 3D59) [START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF] was used as a starting model for molecular replacement minus water molecules and ions. Molecular replacements were carried out using the program MOLREP of CCP4 [START_REF] Ccp4 | The CCP4 Suite: Programs for Protein Crystallography[END_REF] and refinements of each OP complex were accomplished using the program REFMAC5 of CCP4 [START_REF] Ccp4 | The CCP4 Suite: Programs for Protein Crystallography[END_REF]. Model building and modification have were done using the graphics program COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF].

The final molecular replacement solutions consisted of two subunits in the asymmetric unit for sarin, soman and DFP complexes, whereas for the tabun complex there were three molecules per asymmetric unit. Extra difference electron density contiguous with the active site residue Ser273

A c c e p t e d M a n u s c r i p t 11 was identified in 2Fo-Fc difference electron density maps in all four complexes and determined to be the covalently attached OP inhibitor. In all cases there was unambiguous difference electron density of the proper configuration to build in non-aged OP adducts. Since the sarin used was racemic, the resulting inhibited complex could potentially have one or both of two possible stereoisomers (P R and P S ) at the P-chiral center. Soman, which has 2 chiral centers, had the potential to have four stereoisomers (P R C R , P R C S , P S C R and P S C S ) in the crystal structure. We have modeled the P R and P S forms at the P-center of the sarin conjugate with the occupancy of each set to 0.5. And likewise for soman, the P R C S and P S C S forms were modeled with an occupancy of each set to 0.5. The tabun compex was modeled in only the P S conformation, as this form dominated in the crystal structure. For DFP the achiral complex was modeled. Models were refined with the program REFMAC5 [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF]. A CIF parameter file for the non-aged complexes formed after reaction with sarin, soman, tabun, and DFP was prepared using the monomer library sketcher module of the program CCP4 [START_REF] Ccp4 | The CCP4 Suite: Programs for Protein Crystallography[END_REF].

Mass spectrometry of trypsin digests

Although the crystallographic data gave convincing evidence for the presence of non-aged complexes with soman, an additional confirmation of the presence of a non-aged complex was sought via mass spectrometry analysis of peptides formed by trypsin digestion of soman inhibited gVIIA-PLA2 to detect modification at the active site serine (Ser273). Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis was done on soman inactivated complexes of gVIIA-PLA2. Soman was chosen for the mass spectrometry investigation since it is a branched OP that is known to age at a fast rate in complexes with AChE and BChE [START_REF] Masson | Aging of diisopropyl-phosphorylated human butyrylcholinesterase[END_REF]. Peptides were eluted in 20 µl matrix solvent (50% acetonitrile / 0.1% trifluoroacetic acid/1 mM ammonium phosphate), dried completely in a vacufuge and stored at -20 ˚C until ready for analysis by MALDI-MS.

Identification of proteins by MALDI-MS

The dried peptide fragments were resuspended in 2-5 µl matrix solution (2.5 mg/ml α-cyano-4hydroxycinammic acid; Sigma-Aldrich) in matrix solvent (50% acetonitrile/0.1% trifluoroacetic Proteomics Analyzer (Applied Biosystems). Data acquisition by the instrument was controlled with 4000 Series Explorer Software v3.0, acquiring spectra using delayed extraction and reflectron mode with positive ion detection. GPS Explorer Software v3.5 with Mascot was used to identify proteins by peptide mass fingerprinting. Interpretable mass spectra with high sequence coverage were obtained for all peptide fragments.

Analysis of stereoselectivity by reaction with racemic sarin

We studied the stereoselectivity of plasma gVIIA-PLA2 for specificity in binding the stereoisomers of sarin by tandem gas chromatography/mass spectrometry (GC/MS) using methods modified from Yeung et al [START_REF] Yeung | Direct detection of stereospecific soman hydrolysis by wild-type human serum paraoxonase[END_REF]. Assays were performed at the U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Grounds under controlled conditions.

For sarin binding, a 50 µL sample of purified gVIIA-PLA2 (~66 nM) or buffer ( OP compound DFP was used as an internal standard, and its concentration was monitored with three ions (m/z 69, 101 and 127). The total area under the curve was used to develop ratios of each isomer between samples.

Results

Crystal structures of organophosphorus complexes of gVIIA-PLA2

The refined models of OP complexes of gVIIA-PLA2 following inactivation with DFP, sarin, soman and tabun each have a typical α/β lipase fold virtually identical to the previously reported ligand-free and paraoxon bound forms [START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF]. The details of the data collection and structural refinement for all four complexes are summarized in Table 1. The DFP, sarin and soman complexes were solved in the space group C2 with 2 subunits in the asymmetric unit. This packing was isomorphous to the ligand-free and paraoxon bound form previously reported [START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF].

The tabun inactivated complex crystallized in a new space group (C222 1 ) and had 3 subunits in the asymmetric unit. The enzyme is believed to function as a monomer [START_REF] Min | Platelet-activating factor acetylhydrolases: broad substrate specificity and lipoprotein binding does not modulate the catalytic properties of the plasma enzyme[END_REF][START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF]; yet the crystal structures reported here have fairly extensive inter-dimer or inter-trimer interfaces, as shown in Fig. 2 for the soman and tabun complexes, respectively.

The sarin gVIIA-PLA2 complex was modeled with residues 54-425 in subunit A and 54-426 in subunit B; a few residues at the N-terminus (47-53) and C-terminus (426-429 in A and 427-429 in B) were not modeled due to disorder. The soman complex was modeled with residues 54-425 in both subunits and N-and C-terminal residues were not modeled due to disorder. The DFP complex was modeled with residues 54-425 in subunit A, and 54-426 in structures solved to date. There are several surface loop residues in each of the structures that were modeled with alternate conformations. The overall quality of each complex structure is very good as demonstrated by the percentage of residues in most favored and additionally allowed regions of Ramachandran plot (Table 1). A comparison between the ligand-free and OP-complex crystal structures shows an overall C α RMSD of 0.31 Å for DFP, 0.28 Å for sarin, 0.25 Å for soman, and 0.50 Å for tabun, which indicates the structures of OP-complexes are essentially identical to that of the ligand free protein structure [START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF].

DFP complex with gVIIA-PLA2

Sufficient quality difference electron density was present to model in the entire serinephosphonyl diisopropyl covalent species. The model of the DFP complex with gVIIA-PLA2 is highly similar to the complex formed following reaction with the OP paraoxon [START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF] and is presented in supplemental-Fig. 1A. Several interactions between the inhibitor and the enzyme have been observed and are listed in Table 2 for each OP complex presented. The double bonded phospho oxygen is interacting with the oxyanion hole of the enzyme through H-bonds to the backbone amides of Phe274 and Leu153. His351 is seen making a H-bond with the phosphoester oxygen of this substituent where His351 is unable to activate a water molecule for catalytic turnover. Presumably, His351 is protonated as a result of proton abstraction from His351 from reverting to an unprotonated form and activating a water molecule for a nucleophilic attack, which could reactivate the enzyme.

In the DFP complex, one isopropyl group projects toward the solvent channel, which is close to the catalytic His351, and the other isopropyl group is away from histidine; the oxygen to His351 distances are 2.99 and 4.07 Å respectively. None of the oxygen atoms attached to isopropyl groups are accessible to the solvent water and their solvent accessibility becomes almost zero in the complex. Here, the orientation of the alkyl groups have made water inaccessible to the P atom of the OP conjugate as well. Additionally, a lack of water accessibility to His351 suggests the histidine-assisted activation of water for aging is not likely in this complex. Therefore aging through either P-O bond or C-O bond cleavage is not feasible, and the complex is clearly not aged in the crystal structure. The double bonded phosphorusoxygen is interacting with the oxyanion hole of the enzyme through H-bonds to the backbone amides of Leu153 and Phe274. Although the contact distances of the neighboring residues to Ser273 are higher than that observed in the ligand-free structure of the enzyme, other geometrical parameters of neighboring residues are essentially the same in order to maintain the structural integrity of the enzyme.

Sarin complex with gVIIA-PLA2

The model of the gVIIA-PLA2 sarin complex shares many of the interactions discussed above for the DFP complex structure. However, there are a few interesting details worth noting. Sarin has two stereoisomers due to the chiral center at P. There appears to be both stereoisomers of the P-center bound to the enzyme at equal occupancy, and modeled accordingly with an occupancy presented in supplemental-Fig. 1B. When the DFP and sarin complex structures are superimposed, the placement of the isopropyl group of sarin from each stereoisomer (P R and P S ) overlaps the two isopropyl groups which exist in the DFP gVIIA-PLA2 complex. The linker oxygen between Ser and P is invariant and has its occupancy set at 1.0 during refinement.

Likewise, the double bonded phosphorus-oxygen, which is interacting with the oxyanion hole of the enzyme through H-bonds to the backbone amide of Leu153 and Phe274, also maintained an occupancy of 1.0. Therefore, in order to model the two distinct stereoisomers at the P-center (P R and P S ), the methyl and the oxy-isopropyl group attached to P were interchanged and their occupancies were maintained as 0.5 for each alternate conformation. The projection of the oxyisopropyl group in P R stereoisomer was the same as that in the DFP complex structure and that was near the catalytic histidine, whereas the oxy-isopropyl group of the Ps isomer was away from His351 and similar to the second oxy-isopropyl group seen for the DFP complex structure.

The contact distance of the neighboring residues to Ser273, which is attached to the OP, is almost the same as they are in the ligand-free structure.

Soman complex with gVIIA-PLA2

The gVIIA-PLA2 soman complex exhibits many of the features of DFP and sarin complexes.

However, soman has a total of four stereoisomers due to chiral centers at the P, as well as the C α of the pinacolyl group. In the crystal structure it was not possible to refine four different stereoisomer conformations that are likely present, based on our observation of GC mass spectrometry data (see below). In the final structure we have modeled two stereoisomers, the P R C S and P S C S conformation with the occupancy set at 0.5 for each. The presence of P R and P S A c c e p t e d M a n u s c r i p t 18 stereoisomers, shown in Fig. 3A, clearly indicates that the soman complex does not age. As mentioned above for the sarin complex, the solvent accessibility of the conjugate oxygen atoms close to His351 or P is hindered by the large pinacolyl alkyl group. Similar to the sarin complex, the contact distances of the residues neighboring Ser273 in the soman complex are nearly the same as they are in the ligand-free structure.

Tabun complex with gVIIA-PLA2

Unlike the ligand-free enzyme or other OP complexes, the gVIIA-PLA2 tabun crystallized as a trimer in the asymmetric unit. The S N 2 reaction by Ser273 followed by removal of a cyanide leaving group, as shown in Fig. 3B, made this complex chiral with the formation of a P R complex. Despite the inversion at the P-chiral center, the P R adduct is derived from the reaction of P R tabun due to the loss of the CN group and nomenclature rules (Fig. 3B). An interesting change in this structure occurs as the electronic effect of N,N-dimethyl group displaced the active site His351 away from its native and functional conformation. In its present conformation Hbonding is not possible to stabilize the transition state for the aging process. In this non-aged tabun complex extra stability comes from a CH…π interaction between the N,N-dimethyl group and Phe322 and Trp298 of subunit A, and Phe51of subunit C [START_REF] Chakrabarti | CH/pi interaction in the packing of the adenine ring in protein structures[END_REF][START_REF] Samanta | Ab initio study of energetics of X-H center dot center dot center dot pi (X = N, O, and C) interactions involving a heteroaromatic ring[END_REF][START_REF] Nishio | The CH/pi interaction: Nature, evidence and consequences[END_REF]. Like the DFP complex presented above, the contact distances of immediate neighboring residues to Ser273 are higher than those observed in the ligand-free structure of the enzyme. Other geometrical parameters of neighboring residues are essentially the same, thus maintaining the structural integrity of the enzyme.

Mass spectrometry analysis of OP inhibited gVIIA-PLA2

A c c e p t e d M a n u s c r i p t

19 While the electron density maps of the gVIIA-PLA2 soman and sarin complexes were highly suggestive of full occupancy of non-aged inhibitor, analysis via mass spectrometry was pursued as further proof and to ensure that the difference electron density maps were not being misinterpreted. Uninhibited plasma gVIIA-PLA2 along with enzyme inhibited by soman were subjected to trypsin digestion and MALDI-TOF MS analysis. The peptide fragment generated after trypsin digestion containing the active site serine (Ser273) has the predicted sequence 267-IAVIGHSFGGATVIQTLSEDQR-288 with an expected mass of 2299.548 daltons. The peptide was observed in both the untreated and soman inhibited samples, although its abundance was dramatically decreased in the soman inhibited sample. The MALDI mass spectra are presented in supplemental-Fig. 2. Table 3 lists the key detected peptide peaks with masses equal to and greater than the Ser273 peptide. We observed three new peptide masses after soman inhibition, which correspond to the addition of 48.97, 83.96 and 162.10 daltons to the Ser273 peptide. The addition of 162 daltons is the change in mass predicted for the addition of a soman molecule after loss of its fluoride leaving group upon binding. If an OP aging event occurred, we would expect the total mass addition to be only 77 daltons after loss of the secondary alkoxy leaving group. No fragment from the inhibited peptide pool was observed which corresponded with a mass 77 daltons greater than the uninhibited peptide. The presence of a peak consistent with a non-aged adduct at 2461.3303 daltons and the absence of a peak of the size predicted for an aged product further support the identity of a non-aged soman adduct in our complexes. The unmodified plasma gVIIA-PLA2 sample showed a major peak consistent with the predicted mass of an unmodified Ser273, and the soman inhibited protein spectra is likewise consistent with the identity of a non-aged enzyme complex (supplemental-Fig. 2).

A c c e p t e d M a n u s c r i p t 20

Analysis of stereoselectivity by reaction with racemic sarin

We studied stereoselective binding of sarin to gVIIA-PLA2 and ICOS PAFase (Fig. 4) by GC/MS using a chiral column to separate the two stereoisomers of sarin (P S and P R ). Prior experiments that reacted sarin with human BChE have enabled us to assign the second peak as the P S stereoisomer of sarin (not shown). We observed bulk binding of sarin to both ICOS and recombinant gVIIA-PLA2, which was observed by the decrease in the peak area compared to the control of sarin in buffer. There was no stereoselective preference for binding of either sarin isomer. ICOS PAFase was tested in parallel with similar results. This qualitative result is consistent with the crystallographic data, which likewise supports the lack of preference of gVIIA-PLA2 for either stereoisomer of sarin.

Discussion

Although structures of gVIIA-PLA2 inhibited by OPs do not definitively serve in identifying the physiological substrates of the lipoprotein associated enzyme, they do provide clues to important interactions of the putative hydrolase mechanism. Here the OP inhibited structures help to further describe the limits of the possible modes of substrate binding. The previously reported crystal structure of gVIIA-PLA2 inhibited by the OP compound paraoxon [START_REF] Samanta | Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis[END_REF] allowed us to predict how a substrate would access and bind to the active site. This covalent complex served as a tetrahedral intermediate mimic of the esterolysis reaction, and allowed us to substitute the paraoxon derived diethyl phosphate conjugate from our crystal structure with a speculative model of the tetrahedral intermediate of the physiological PLA2 reaction of phospholipid with a short sn-2 chain. It was predicted that the larger lyso-moiety, which would be the leaving group from the collapse of the tetrahedral intermediate, projects out of the active site toward the solvent A c c e p t e d M a n u s c r i p t 21 and polar portion of the interface. A smaller pocket projects inward that has been modeled with the sn-2 chain of the substrate. Although this pocket may limit the size of the sn-2 chain of substrates, and thereby explain the enzyme's preference for a shortened and polar sn-2 chain [START_REF] Davis | Electrospray ionization mass spectrometry identifies substrates and products of lipoprotein-associated phospholipase A(2) in oxidized human low density lipoprotein[END_REF][START_REF] Min | Membranebound plasma platelet activating factor acetylhydrolase acts on substrate in the aqueous phase[END_REF][START_REF] Min | Platelet-activating factor acetylhydrolases: broad substrate specificity and lipoprotein binding does not modulate the catalytic properties of the plasma enzyme[END_REF], it is a fairly open pocket that could accommodate larger chains. Our structures of gVIIA-PLA2 complexed with conjugates derived from DFP, sarin and soman reinforce this model. The DFP complex structure has two isopropyl groups, which overlay where the diethyl groups of the paraoxon complex were modeled. The preference of each of these pockets to bind an isopropyl group is better illuminated by the racemic nature of binding in the sarin complex. Here the isopropyl group has adopted a nearly 50/50 distribution between these two pockets, and the smaller methyl group of sarin does as well. When you compare the DFP and sarin complex structures (supplemental-Fig. 1), the binding of these isopropyl groups into both of these pockets would be the expected result. Furthermore, the racemic binding of the soman conjugate (Fig. 3)

shows that a larger pinacolyl group binds to each of the two pockets with nearly equal affinity.

The observation of fully interpretable electron density difference maps (coefficients 2Fo-Fc) for both isopropyl groups of DFP, with the occupancy set at 1.0, is evidence that the complex does not go through the aging process, which would lead to the P-O bond cleavage of one of the isopropyl groups. Likewise, a resistance of the OP conjugate to aging was observed by the racemic modeling of sarin and soman, modeled at 0.5 occupancy in alternate conformations with isopropy and pinacolyl groups, respectively. The stability of the non-aged conjugates of sarin, soman and tabun was demonstrated by the long incubation period of protein co-crystallization of 4-7 weeks at 20 ˚C. The DFP complex crystal was formed by soaking pre-grown crystals for ~20 min. Further support for a stable non-aged complex of the soman conjugate was shown by MALDI-MS of trypsin digests of soman-inhibited gVIIA-PLA2 enzyme. When samples were A c c e p t e d M a n u s c r i p t 22 compared pre and post treatment with soman, a peptide containing Ser273 was shifted to a mass consistent with a non-aged complex with soman. The decrease of the unmodified peak, as well as the lack of a peak consistent with an aged conjugate, strengthens our conclusion that the soman complex did not show any detectable aging reaction.

Many serine hydrolases have been demonstrated to promote the aging reaction when reacted with branched OPs, such as trypsin with DFP [START_REF] Kossiakoff | [END_REF], or AChE complexes with sarin, soman or DFP [31]. Each of these enzymes shares the common element of a catalytic histidine to carry out proton transfer and a solvent accessible active site. Recently two enzymes have been shown to be resistant to aging of branched OP complexes; non-aged crystal structures have been reported of human carboxylesterase complexed to soman and tabun [START_REF] Fleming | Crystal structures of human carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun[END_REF], and gVIII-PLA2 complexed with sarin and soman [START_REF] Epstein | Crystal structures of brain group-VIII phospholipase A2 in nonaged complexes with the organophosphorus nerve agents soman and sarin[END_REF]. A comparison of the enzyme complexes reported here to both enzymes that have rapid OP aging, as well as those that do not, is instructive to understand the determinants of aging. The most notable explanations of rapid aging rates in the AChE enzyme are due to i) the mobility of the histidine in the AChE enzyme has been argued to be critical to explain its broad specificity and rapid rates of reaction [START_REF] Millard | Reaction Products of Acetylcholinesterase and VX Reveal a Mobile Histidine in the Catalytic Triad[END_REF]; ii) a cation-π interaction of Trp86 in AChE is believed to stabilize a carbocation intermediate [START_REF] Ordentlich | Exploring the active center of human acetylcholinesterase with stereomers of an organophosphorus inhibitor with two chiral centers[END_REF]; and iii) the oxygen adjacent to the developing carbocation intermediate of the aging reaction is at least partly water accessible. When one compares the gVIIA-PLA2 enzyme to AChE under these criteria the following observations can be made. The histidine may not have the same degree of mobility as in the AChE enzyme. The OP complexes with DFP, sarin and soman show the histidine to be restricted in movement by the surrounding residues and the alkyl groups of the bound OP conjugate. The complex formed by reaction with tabun was different, and in this case His351 was completely displaced away from a mechanistically significant position (Fig. 3B). Next, the However, the geometric positioning of this interaction is not identical and may preclude its utility. This positioning may be critical, as aging rates have been previously rationalized based on the ability of the developing carbocation to be directly stabilized by the enzyme active site [START_REF] Barak | Carbocation-mediated processes in biocatalysts. Contribution of aromatic moieties[END_REF]. Therefore, it is reasonable to assume that the dielectric of the surroundings and the presence or absence of cation-π interactions would be a factor in the ability of the carbocation to form and be stabilized. The CH…π interaction between the methyl groups of the N,N-dimethyl moiety of tabun and the side chains of Phe322, Trp298 of subunit A, and Phe51 of subunit C bring extra stability to the non-aged tabun complex. Finally, the binding of DFP, sarin, soman or tabun to gVIIA-PLA2 results in the placement of hydrophobic alkyl groups in positions that make the functionally significant active site atoms virtually inaccessible to water. The alkyl groups of the conjugates block an incoming water molecule and restrict any interaction with His351 as well. The exclusion of water from the active site in gVIIA-PLA2 complexes might be the key to understanding the lack of aged adducts. While it is known that for AChE and BChE, aging is an enzyme catalyzed process, it is slow for most OP compounds compared to other enzyme-mediated processes. In light of the consensus mechanism for aging (Fig. 1), the current results are not surprising, since a carbocation must be formed by charge separation. Therefore, it would be reasonable to assume the change of dielectric of the surroundings would be a factor in the ability to form the carbocation. The more solvent accessible active sites of chymotrypsin, AChE and BChE yields a higher dielectric in their active sites, thus enabling these active sites to be reactivated by the attack of activated nucleophiles on their OP adducts. In contrast, gVIIA- In the present work, gVIIA-PLA2 has been identified as an additional enzyme that forms stable non-aged complexes of nerve agents, such as DFP, sarin, soman and tabun. The enzymes carboxylesterase and gVIII-PLA2 are two other systems that have been shown to be resistant to aging, but each displays greater stereoselectivity for the nerve agents than does gVIIA-PLA2.

Human carboxylesterase was shown to react preferentially with the less toxic P R stereoisomer of soman and the P S stereoisomer of tabun [START_REF] Fleming | Crystal structures of human carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun[END_REF]. AChE reacts preferentially with the P S stereoisomer of soman [START_REF] Benschop | Isolation, anticholinesterase properties, and acute toxicity in mice of the four stereoisomers of the nerve agent soman[END_REF], and the P R stereoisomers of tabun [START_REF] Ekstrom | Crystal structures of acetylcholinesterase in complex with HI-6, Ortho-7 and obidoxime: Structural basis for differences in the ability to reactivate tabun conjugates[END_REF]. The gVIII-PLA2 enzyme was previously shown to react with the more toxic P S stereoisomers of sarin and soman [START_REF] Epstein | Crystal structures of brain group-VIII phospholipase A2 in nonaged complexes with the organophosphorus nerve agents soman and sarin[END_REF]. In the gVIII-PLA2 system, a crystal structure and GC/MS analysis of the sarin reaction gave convincing evidence of stereoselective preference of reaction [START_REF] Epstein | Crystal structures of brain group-VIII phospholipase A2 in nonaged complexes with the organophosphorus nerve agents soman and sarin[END_REF]. This contrasts our present work, where we have shown that gVIIA-PLA2 reacts non-selectively with both P R and P S stereoisomers of sarin and soman. In contrast, the enzyme showed stereospecificity for the more toxic P R stereoisomer of tabun. Together, the nascent reactivity of gVIIA-PLA2 with sarin, soman and tabun serves as a starting point to engineer the enzyme to display OP hydrolase activity, which has already enjoyed limited success in other serine hydrolases [START_REF] Lockridge | A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase[END_REF][START_REF] Millard | Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase[END_REF]. This approach contrasts with other catalytic bioscavengers in development that utilize a mechanism in which the initial attack is by a catalytic water molecule [START_REF] Amitai | Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase[END_REF]. However, despite notable catalytic efficiencies, these enzymes typically have high K M values; therefore they suffer from an inability to efficiently bind OP compounds at concentrations shown to be lethal in test animals. The structural characterization of the interactions between the oxyanion hole of gVIIA-PLA2 and the OP adducts of DFP, sarin, soman and tabunallow the rational development of a catalytic A c c e p t e d M a n u s c r i p t 25 hydrolase with a lower K M value than enzymes like paraoxonase [START_REF] Amitai | Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase[END_REF] or phosphotriesterase [START_REF] Ghanem | Detoxification of organophosphate nerve agents by bacterial phosphotriesterase[END_REF].

The versatility of the gVIIA-PLA2 active site is currently being exploited to generate, through mutagenesis, catalytic bioscavengers of highly toxic OPs that will work at the physiologically relevant and low concentrations of OPs that are known or estimated to be toxic in vivo. R free is the equivalent of R working , except it is calculated for a randomly chosen set of reflections that were omitted (5%) from the refinement process [START_REF] Brunger | The free R value: A novel statistical quantity for assessing the accuracy of crystal structures[END_REF]. Values shown in the parenthesis are in the highest resolution shell.

A c c e p t e d M a n u s c r i p t 33

A c c e p t e d M a n u s c r i p t 10 inhibited

 10 protein were flash frozen directly from the crystallization drop into liquid nitrogen, which allowed facile transport to the synchrotron beam line. Crystals were flash cooled without the need of an additional cryo-protectant. The treatment of DFP inhibited protein crystals varied.

A c c e p t e d M a n u s c r i p t 12 Inhibition

 12 of gVIIA-PLA2 with soman was performed at the U.S. Army Medical Research Institute of Chemical Defense as described above for the inhibition of protein crystals.The inhibition of gVIIA-PLA2 was accomplished by adding 1 µL of a 10 mM stock of soman in saline to 10 µL aliquots (0.7 mg/ml) of gVIIA-PLA2 in 10 mM Tris, pH 8. Inhibition of gVIIA-PLA2 with soman was relatively slow compared to the cholinesterases, and was confirmed by assaying enzyme activity using the substrate p-nitrophenylacetate. Inhibited gVIIA-PLA2 was incubated at 4 ˚C for at least 48 hours before proceeding to trypsin digestion.Samples were buffer exchanged into 50 mM ammonium bicarbonate using BioGel P-4 micro spin buffer exchange columns (BioRad) to remove any residual OP compound that could interfere with trypsin digestion. All samples, including an uninhibited control sample, were separated by SDS PAGE electrophoresis, stained with 0.1% Coomassie blue, and isolated by excision of selected protein bands. In-gel digestion was performed using Zip Plates with the MultiScreen HTS vacuum manifold (Millipore, Billerica, MA). Each gel piece was washed, dried, reduced by treatment with 10 mM DTT and alkylated by treatment with 55 mM iodoacetamide (Sigma-Aldrich). The treated gel spot was dried and rehydrated in 25 mM ammonium bicarbonate containing 11 ng/µl trypsin (Pierce) and incubated at 37 ˚C overnight.

  A c c e p t e d M a n u s c r i p t 13 acid/1 mM ammonium phosphate). The resuspended peptides were spotted on a stainless steel target plate. The sample was analyzed using a MALDI tandem Time-of-Flight (TOF-TOF) 4700

  A c c e p t e d M a n u s c r i p t 14 5973 mass spectrometer with an electronic impact ion source under selected ion monitoring mode. The mass to charge (m/z) peaks at 99 and 125 were monitored for sarin quantitation. The

A c c e p t e d M a n u s c r i p t 15 subunit

 15 B. The tabun complex was solved from a different space group, which contained 3 subunits per asymmetric unit. Additionally, an unusual CH…π interaction between the N,Ndimethyl group of the tabun conjugate in subunit A and Phe51 of subunit C increased the order of the N-terminal resides. The tabun complex was more ordered than the other complexes, with five additional residues modeled at the N-terminal compared to each of the other gVIIA-PLA2

A c c e p t e d M a n u s c r i p t 16 Ser273

 16 concurrent with nucleophilic attack on DFP. This direct H-bond interaction precludes

A c c e p t e d M a n u s c r i p t 17 of 0

 170 .5 during refinement of the structure. The racemic model of sarin bound to gVIIA-PLA2 is

A c c e p t e d M a n u s c r i p t 23 active

 23 site of gVIIA-PLA2 contained a tryptophan residue analogous to Trp86 in AChE, which could provide the critical cation-π interactions with a developing carbocation intermediate.

A c c e p t e d M a n u s c r i p t 24 PLA2

 24 branched OP complexes are positioned with the alkyl chain blocking solvent access, and as a result lowering the local dielectrics surrounding key active site atoms.

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 29 Figure Legends Fig. 1 -

 29Legends1 Figure Legends

Fig. 2 -

 2 Fig. 2 -Surface and ribbon diagram of the soman-dimer and tabun-trimer crystal structure

Fig. 3 -

 3 Fig. 3 -The reaction of gVIIA-PLA2 with soman and tabun to Ser273 are shown relative to

Fig. 4 -

 4 Fig. 4 -Analysis of the reaction of gVIIA-PLA2 with sarin. The control (top black trace) is an

a

  R merge = Σ|I o -I a |/Σ(I a ), where I o is the observed intensity and I a is the average intensity, the sums being taken over all symmetry related reflections. b R-factor = Σ|F o -F c |/Σ(F o ), where F o is the observed amplitude and F c is the calculated amplitude.

A c c e p t e d M a n u s c r i p Figure 1 Page 36 of 38 A c c e p t e d M a n u s c r i p t

 138 Figure 1

Figure 2 Page 37 of 38 A c c e p t e d M a n u s c r i p t

 238 Figure 2

Figure 3 A c c e p t e d M a n u s c r i p

 3 Figure 3

Figure 4

 4 Figure 4

  inhibition of a serine hydrolase with an OP compound, such as O-isopropyl methylphosphonofluoridate (sarin, GB) can stop at a non-aged complex, proceed to an "aged" complex through a carbocation mechanism (top branch), or be hydrolyzed to a harmless product (bottom branch). The aging path is promoted by a secondary or tertiary C α , which stabilizes the developing positive charge. The hydrolysis path is promoted, if aging is not fast, and if a nucleophilic water can approach the non-aged species. (B) The OP compounds sarin, O-

pinacolyl methylphosphonofluoridate (soman, GD) and O,O-diisopropylfluorophosphate (DFP) have a branched side-chain that facilitates aging, whereas O,O-diethyl-O-nitrophenylphosphate (paraoxon) and ethyl N,N-dimethylphosphoramidocyanidate (tabun, GA) do not.

Table 1 -Structure determination of OP complexes of gVIIA-PLA2.

 1

	Data set	Sarin	Soman	Tabun	DFP

Table 2 -OP complex contacts within 4Å.

 2

	Residue/atom	DFP	Sarin a	Soman a	Tabun
	type	contact atom,	contact atom,	contact atom,	contact atom,
		distance (Å)	distance (Å)	distance (Å)	distance (Å)
	F51C, CZ b	-	-	-	C2, 3.47
	G152A, CA	O3P, 3.17	O1, 3.54	C3, 3.13	O1, 3.37
	L153A, N c	O3P, 2.57	O1, 2.65	O11, 2.76	O1, 2.62
	A155A, CB	-	-	C3, 3.78	-
	Y160A, OH	C3', 3.75	-	C3, 3.34	C4, 3.94
	H272A, CE1	-	C4, 3.70	C2, 3.20	C4, 3.78
	F274A, N	O3P, 2.65	O1, 2.74	O11, 2.97	O1, 2.72
	W298A, CE3	-	C1, 3.71	-	C1, 3.68
	F322A, CE1	-	-	-	C2, 3.68
	H351A, NE2	O2P, 2.99	O2, 2.91	OH, 3.11	O2, 3.53
	Q352A, NE2	C2', 3.51	C3, 3.98	C2, 2.91	C4, 3.69

a For PsCs stereoisomer of soman and Ps stereoisomer of sarin.

b Residues are defined with single letter code, residue number and subunit relative to OP complexes in the A-subunit of each structure.

c Hydrogen bond contacts are shown in bold italics.

Acknowledgements

We thank ICOS Corp. for supplying us with the protein PAFase. We thank Dr. James Dillman and Ms. Lisa Bottalico for obtaining MALDI-MS data and Mr. Rick Smith for helping to complete the GC/MS analysis at the USAMRICD. We also thank the support staff of 19ID and 24ID beam lines of Advance Photon Source at the Argonne National Laboratory for assistance during X-ray diffraction data collection.

sarin, soman and tabun, respectively. This work was supported by NIH Grants 2P20RR015588 from the National Center for Research Resources and 1R01HL084366-A1 from the National Heart, Lung, and Blood Institute.