Vol. 173, No. 6

Purification and Properties of an Organophosphorus Acid Anhydrasefrom a Halophilic Bacterial Isolate

JOSEPH J. DEFRANK* AND TU-CHEN CHENG

U.S. Army Chemical Research, Development & Engineering Center, Biotechnology Division, Research Directorate,Aberdeen Proving Ground, Maryland 21010-5423

Received 18 October 1990/Accepted 14 January 1991

A moderately halophilic bacterial isolate has been found to possess high levels of enzymatic activity againstseveral highly toxic organophosphorus compounds. The predominant enzyme, designated organophosphorusacid anhydrase 2, has been purified 1,000-fold to homogeneity and characterized. The enzyme is a singlepolypeptide with a molecular weight of 60,000. With diisopropylfluorophosphate as a substrate, the enzyme hasoptimum activity at pH 8.5 and 50°C, and it is stimulated by manganese and cobalt.

Organophosphorus acid (OPA) anhydrases are enzymesthat are capable of catalytically hydrolyzing a wide variety oforganophosphorus cholinesterase inhibitors, among themdiisopropylfluorophosphate (DFP), the chemical warfareagents soman (0-1,2,2-trimethylpropyl methylphosphonof-luoridate), sarin (O-isopropyl methylphosphonofluoridate),and tabun (ethyl N,N-dimethylphosphoramidocyanidate),and the pesticides parathion (diethyl p-nitrophenyl phospho-rothioate) and paraoxon (diethyl p-nitrophenyl phosphate)(10, 12, 13). Enzymes such as these are of interest for theirpotential use in decontamination and demilitarization ofthese extremely toxic materials. In the past, these enzymeswere known variously as DFPases, somanases, parathionhydrolases, or paraoxonases, depending on the assay sub-strate used. Sources of these enzymes range from bacteriaand protozoans to higher mammals, including humans, andthe number of enzymes found has greatly increased in recentyears (10). The proliferation of both enzymes and enzymenames led to the adoption of the name OPA anhydraseduring the First DFPase Workshop (Marine Biological Lab-oratory, Woods Hole, Mass., June 1987) to describe theserelated enzymes. It was planned that this name be used untilthe natural substrates and functions of these enzyme areidentified. Preliminary studies of OPA anhydrases fromvarious sources have demonstrated that these enzymes differin substrate specificity, sensitivity to inhibitors, activationby metals, and molecular weight (10). Purification and char-acterization of these enzymes, such as the one described inthis report, may assist in the determination of the true natureof their substrates, specificity, and molecular structure.The source of the enzyme to be discussed is the obligately

halophilic bacterial isolate designated JD6.5, which wasisolated from a warm salt spring. This isolate was found topossess high levels of DFP-hydrolyzing OPA anhydraseactivity (3). In this report we describe the purification andcharacterization of OPA anhydrase 2 (OPAA-2), the pre-dominant enzyme from JD6.5.

MATERIALS AND METHODS

Organism and cultivation. Isolate JD6.5 was obtained fromGrantsville Warm Springs, which is located approximately30 miles (ca. 48 km) west of Salt Lake City, Utah, and justsouth of the Great Salt Lake. The primary characteristics of

* Corresponding author.

the springs are a relatively constant temperature of 24°C, a

pH of 6.0, and a salt content of approximately 24% (14).Cultures were grown in a medium consisting of the following(grams per liter): NaCl, 50; MgSO2 7H20, 10; ProteosePeptone (Difco), 10; yeast extract, 6; and N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid (HEPES), 2.5 (pH6.8). Inoculated flasks (4 or 6 liter) containing 1 to 1.5 litersof medium were incubated at 30 to 37°C, on a rotary shakerat 240 rpm, for 18 to 24 h. Cells were harvested by centrif-ugation (7,500 x g) at 20°C (to prevent precipitation of an

unidentified saltlike material observed at lower tempera-tures) and stored at -20°C.Enzyme assays. OPA anhydrase activity was routinely

assayed by monitoring fluoride release from DFP by an

ion-specific electrode as has been described numerous timesin the literature (6, 7, 11). Unless otherwise stated thereaction medium contained 500 mM NaCl, 50 mM Bis-Trispropane (pH 7.2), 3.0 mM DFP, 1.0 mM MnCl2 4H20, and5 to 25 jil of enzyme sample in a total volume of 2.5 ml. ThepH of 7.2 was selected to be the standard for all assays inorder to be consistent with the numerous published reportson other OPA anhydrases (5-7, 10-13). Assays were run at25°C in a temperature-controlled vessel with stirring. Theenzyme sample was preincubated in the reaction medium for1 min before the reaction was initiated by the addition ofDFP (0.3 M in isopropanol). The reaction was monitored for4 min, and the rate of fluoride release was corrected forspontaneous DFP hydrolysis under identical conditions. Oneunit of OPA anhydrase activity is defined as catalyzing therelease of 1.0 ,umol of F- per min. Specific activity isexpressed as units per milligram of protein.The hydrolysis of chromogenic substrates was conducted

in a reaction mixture identical to that described above exceptthat the substrate concentration was reduced to 5 to 100 puM.Activity was determined by monitoring the increase inabsorbance at 405 nm (for p-nitrophenol), and units are

expressed as 1.0 ,umol of p-nitrophenol released per min.The concentration of p-nitrophenol was determined from astandard curve with authentic material.Enzyme purification. All procedures were conducted at

4°C, and all centrifugations were at 46,000 x g for 30 min.Frozen or freshly harvested cells from 10 liters of culturewere resuspended in 10BM buffer (10 mM Bis-Tris propane,0.1 mM MnCl2 [pH 7.2]) at a ratio of 3 ml of buffer for eachgram (wet weight) of cells. The cells were disrupted bypassage through a French pressure cell (SLM Aminco) at

1938

JOURNAL OF BACTERIOLOGY, Mar. 1991, p. 1938-19430021-9193/91/061938-06$02.00/0Copyright C) 1991, American Society for Microbiology

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0

0

00c2

4)-

oo

L cU

0.z

0 1 00 200 300

E

c

M-

0

4

Fraction No.

FIG. 1. Separation of OPAA-1 and OPAA-2 by DEAE-Sephacelchromatography. Conditions are described in the text. Symbols:protein; --, NaCl; 0, OPA anhydrase activity.

16,000 lb/in2. Cellular debris was removed by centrifugation.The crude cell supernatant, which contained the OPA anhy-drase activity, was treated with protamine sulfate to a finalconcentration of 0.4% in order to remove nucleic acids andassociated proteins. After centrifugation, the supernatantwas fractionated with solid (NH4)2SO4 to give the 30 to 65%(saturation) precipitate. The pellet was resuspended in a

minimal volume of 10BM buffer and dialyzed against severalchanges of 20 volumes of the same buffer.The dialyzed sample was applied to a DEAE-Sephacel

(Pharmacia) column (5 by 20 cm) previously equilibratedwith 10BM buffer. The column was washed with 10BM toremove nonbinding materials. After the washing, the elutionbuffer was stepped to 200 mM NaCl in 10BM. The OPAA-2activity was eluted with a 4-liter linear gradient of 200 to 600mM NaCl (Fig. 1). Active fractions (193 to 212) were pooled,concentrated by precipitation at 65% (NH4)2SO4, and thencentrifuged. The pellet was dissolved in 10 mM Bis-Trispropane-100 mM NaCl-5 mM KH2PO4 (pH 7.2). The solu-tion was dialyzed overnight against 10 liters of this buffer.The enzyme solution was loaded onto a hydroxyapatite

(HA-Ultrogel; IBF Biotechnics) column (2.6 by 14 cm)previously equilibrated with the Bis-Tris propane-NaCl-KH2PO4 (pH 7.2) buffer described above. (Manganese wasnot used during this chromatographic procedure to preventprecipitation of MnPO4.) After nonbinding protein was re-moved by washing, elution was carried out with a lineargradient of 5 to 150 mM KH2PO4 (Fig. 2). Enzyme fractions(52 to 58) were again pooled and concentrated by 65%

0

C'Ua_ _

._

O&- .)0 0, -

E

0

.4

Fraction No.

FIG. 2. Purification of OPAA-2 on HA-Ultrogel. Conditions aredescribed in the text. Symbols: -, protein; --, phosphate; 0, OPAanhydrase activity.

(NH4)2SO4. After centrifugation, the pellet was redissolvedin 10BM, supplemented with 10 mM NaCl (pH 7.2), anddialyzed against 6 liters of this buffer.The enzyme solution was further purified on an HPLC

(high-performance liquid chromatography)-GTi system(LKB) using two GF-250 columns (0.94 by 25 cm; DuPont) inseries and run with the 10BM-10 mM NaCl (pH 7.2) buffer ata flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected.The pooled enzyme fractions were concentrated with a

Centricon-30 concentrator (Amicon) and loaded onto a 7%polyacrylamide gel. Electrophoresis was performed accord-ing to Laemmli (9) but without sodium dodecyl sulfate (SDS)and dithiothreitol (DTT). Immediately after the electropho-resis, the bands containing enzyme activity were cut out ofthe gel and eluted into the same electrophoresis buffer withan Extraphor electrophoretic concentrator (LKB). The re-sults of these purification procedures are summarized inTable 1.SDS-PAGE and protein determination. SDS-polyacryl-

amide gel electrophoresis (PAGE) was performed accordingto Laemmli (9). Protein samples were boiled in loadingbuffer, in the presence or absence or DTT, prior to running.The protein bands were visualized with the Gelcode silverstain kit (Pierce). The Coomassie protein assay reagent(Pierce) was used for determination of protein concentra-tions, with bovine serum albumin as the standard.

Preparation of polyclonal and monoclonal antibodies. Thepolyclonal antiserum to OPAA-2 was prepared by immuniz-ing a rat (Sprague-Dawley) with the HPLC-purified enzyme

TABLE 1. Summary of purification of OPAA-2 from JD6.5

Purification step Vol Protein Activity Sp act Purification Yield(ml) (mg) (U) (U/mg) (fold) (%)

Crude extract 306.0 3,005.0 801.0 0.267 100.0Protamine sulfate 328.0 2,938.0 853.0 0.290 1.1 106.5Ammonium sulfate 145.0 1,167.0 992.0 0.850 3.2 123.8DEAE-Sephacel 19.0 19.4 570.0 29.38 110.0 71.2HA-Ultrogel 1.3 0.8 205.0 259.49 971.9 25.6HPLC-GF 250 2.0 0.4 143.0 357.50 1,337.1 17.9PAGE 4.0 0.1 26.8 268.00 1,003.7 3.3

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1940 DEFRANK AND CHENG

Af

5w

:_:a

3X

MW

- 200,000

MW B200,000

200,000-

97,000

97,000 97,000- 68,000

68, 000_

43,000

- 43,00043,000-

25,700-6 7 8

MW A

1 2 3 4

B

1 2 3 4

FIG. 4. Representation of Western blot with monoclonal (A) andpolyclonal (B) antibodies. For both blots, lanes 1 to 4 representcrude extract, pooled DEAE-Sephacel fractions (OPAA-1), pooledDEAE-Sephacel fractions (OPAA-2), and purified OPAA-2, respec-

1 2 3 tively. MW, Molecular weight standards.

FIG. 3. (A) SDS-PAGE of fractions from JD6.5 OPAA-2 purifi-cation. Lanes 1 to 8 contain crude extract, protamine sulfatesupernatant, ammonium sulfate pool, pooled DEAE-Sephacel frac-tions, pooled HA-Ultrogel fractions, HPLC fractions, preparativePAGE-purified OPAA-2, and molecular weight (MW) standards,respectively. (B) SDS-PAGE of purified OPAA-2 under reducing(lane 2) and nonreducing (lane 3) conditions. Lane 1 containsmolecular weight standards.

fraction. The rat was first given a footpad injection of 100 ,ugof enzyme in complete adjuvant (Bacto). Three weeks laterit received a subcutaneous booster of 20 ,ug in incompleteadjuvant (Bacto). One week later, a prefusion dose of 5 ,ug insterile saline was administered intravenously. The with-drawal of polyclonal serum and the fusion procedure werestarted 3 days after the final injection. Serum was obtainedby tail bleeding. Spleen cells from the immunized rat wereremoved and fused with mouse myeloma cell line SP2/0Ag14(8). The hybridoma cell clone was detected by enzyme-linked immunosorbent assay, using microtiter plates coatedwith a crude enzyme preparation. Biotinylated rabbit anti-ratimmunoglobulin G conjugated to horseradish peroxidase (ratExtravidin staining kit; Sigma) was used for the detection ofantibodies against OPAA-2. For Western immunoblottinganalysis, the same detection kit was used as in the immun-odetection assay.

RESULTS

Organism. Isolate JD6.5 is a gram-negative, aerobic, shortrod and an obligate halophile that requires at least 2% NaClfor growth (2). Fatty acid analysis (Microbial ID, Newark,Del.) has tentatively identified JD6.5 as a species of Altero-monas, but not the haloplanktis or putrefaciens species thatwere in the data base.

Purification of OPAA-2. The enzyme preparations at dif-ferent stages in the purification process (Table 1) were

analyzed by SDS-PAGE (Fig. 3A). The purification protocoldescribed above yielded an enzyme preparation that ap-

peared to be homogeneous, as judged by a single band witha molecular weight of approximately 60,000. In a second gelanalysis (Fig. 3B), no difference was observed in the elec-trophoretic mobility of purified OPAA-2 under reducing or

nonreducing conditions. The results strongly suggest that thefinal purified enzyme is a single polypeptide.The purified OPAA-2, in addition to the pooled fractions 1

and 2 from the DEAE-Sephacel step and crude extract, wasalso analyzed by Western blotting after SDS-PAGE. Theblots were analyzed with either monoclonal antibody 6 (Fig.4A) or polyclonal antiserum (Fig. 4B). A single band ofpurified OPAA-2 was detected on both blots (lanes 4). Themonoclonal antibody was also shown to react with twoprotein bands (molecular weights of 78,000 and 74,000) inboth the crude extract and pooled fraction 1 (Fig. 4A, lanes1 and 2). These proteins presumably make up the OPAA-1peak observed in Fig. 1. In the blot reacted with antiserum,these two bands and a third band with an estimated molec-ular weight of 53,000 were also detected (Fig. 4B, lanes 1 and2). The results suggest that these three protein bands are

separated from OPAA-2 during the DEAE-Sephacel chro-matography. The fact that both the monoclonal antibody andantiserum react more strongly to OPAA-1 than OPAA-2suggests two possible explanations. OPAA-1 may be theprecursor(s) for OPAA-2 and produced at high concentra-tions but with low specific activity against DFP. Alterna-tively, these proteins may all be descended from a commonancestral protein and still retain antigenic similarity whilepossibly differing significantly in specificity.To characterize the antiserum in terms of biological activ-

ity, the enzyme activity of purified OPAA-2 was determinedafter reaction with dilutions of the antiserum. In thesepreliminary experiments, the purified enzyme (250 ng) wasincubated with dilutions of the antiserum in 25 ,ul of 10BMbuffer at room temperature for 5 min prior to being assayedwith DFP under standard conditions. At a 250-fold dilutionof antiserum, a 50% inhibition of enzyme activity wasobserved (Table 2).

Substrate specificity. The specific activities of OPAA-2against a variety of substrates is summarized are Table 3.Because of the greater sensitivity of the spectrophotometricassay, lower concentrations of chromogenic substrates wereused. Of the substrates tested, the highest activity was withDFP. The enzyme also exhibited activity against two chro-mogenic compounds, p-nitrophenylmethyl(phenyl)phosphi-nate (NPMPP) and p-nitrophenylethyl(phenyl)phosphinate

I-~

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OPA ANHYDRASE FROM A HALOPHILE 1941

TABLE 2. Inhibition of OPAA-2 by antiserum

% ActivitySerum dilution Antiserum Normal rat serum

+ OPAA-2 + OPAA-2

1:100 44.4 105.01:250 49.3 INDa1:500 60.6 103.01:1,000 71.3 100.01:2,000 83.0 98.2Control, no serum 1.0b 1.0bControl, no enzyme ND <O.lC

a ND, Not determined.b Arbitrarily set at 100lo.C 1:100 dilution.

(NPEPP). Paraoxon was hydrolyzed by the purified enzymeat about 3 to 4% the rate of DFP. These results are stillpreliminary, and additional parameters such as Ki,m Vmax,and pH effect for each of these substrates remain to bedetermined.

In addition to these compounds, a variety of potentialsubstrates for esterases, phosphatases, phosphdiesterases,phosphotriesterases, and phospholipases were examined aspotential substrates. These compounds showed little or noactivity with OPAA-2. Mipafox (N,N'-diisopropyl phospho-rodiamidofluoridate), which is only slightly, if at all, hydro-lyzed by OPAA-2, exhibited a significant level of inhibitionof the enzyme for DFP hydrolysis. Figure 5 shows the effectof incubation of OPAA-2 with Mipafox. In this assay,Mipafox was added to the reaction mixture during thepreincubation period before the addition of DFP. At 3.0 mMMipafox, the DFP hydrolysis was inhibited by greater than90%. As has been demonstrated with OPA anhydrases fromhog kidney and Escherichia coli, this inhibition is competi-tive and reversible (5). Dialysis of the inhibited enzymeovernight against 10BM buffer containing 100 mM NaClresulted in complete restoration of activity against DFP (1).

Effect ofpH and temperature. With DFP as a substrate, theeffect of pH on the activity of OPAA-2 was examined; inaddition, the kinetic parameters (4) were determined at eachpH value (Fig. 6). The apparent pH optimum for activity(Kcat) for OPAA-2 was found to be 8.5. The highest level ofcatalytic efficiency (KcatlKm) was observed at pH 6.8. Thisvalue reflects the pKa of the enzyme and the identity of

TABLE 3. Substrate specificities of OPAA-2a

Substrate ~Concn % ActivitySubstrate ((mM) -Manganese +Manganese

DFP 3.0 71.3 100.0bNPMPP 0.1 35.1 49.2NPEPP 0.1 28.3 39.7Paraoxon 0.1 2.8 3.9Mipafox 1.0 <1.0 <1.0p-Nitrophenyl acetate 0.1 <1.0 <1.0p-Nitrophenyl phosphate 0.1 <1.0 <1.0Bis(p-nitrophenyl) phosphate 0.1 <1.0 <1.0Tris(p-nitrophenyl) phosphate 0.1 <1.0 <1.0p-Nitrophenyl(phenyl) phosphonate 0.1 <1.0 <1.0p-Nitrophenyl phosphorylcholine 0.1 <1.0 <1.0

a Assay conditions are described in the text.b Activity of HPLC-purified OPAA-2 with DFP plus 0.1 mM manganese set

at 100%. This value corresponds to a specific activity of 357.5 U/mg.

>b 60

40-

20-

0 I

0.0 1.0 2.0 3.0

Mlpafox [mM]

FIG. 5. Inhibition of OPAA-2 by preincubation with increasingconcentrations of Mipafox. Conditions are described in the text.

ionizing groups in the active site or on the surface. Fromthese observations, the presence of a histidine or a cysteineat its catalytic site is suggested (4).The effect of temperature on the reaction rate of the

hydrolysis of DFP by OPAA-2 was examined. The initialreaction rate of the enzyme with 3.0 mM DFP, at pH 7.2,reached its maximum at 50°C in the presence of 1.0 mMMnCl2. The level of activity decreased at 55°C, which wasthe highest temperature tested in order to protect the fluorideelectrode. The purified enzyme could be stored for months at-70°C in the presence ofDTT. The addition ofDTT not onlyimproved stability but appeared to stimulate activity over

pHFIG. 6. Effect of pH on OPAA-2 hydrolysis of DFP and kinetic

parameters. Units: Kcat seconds-' or micromoles per minute permilligram; Kcat/Ki,m seconds-' molar concentration-1; Ki,, millimo-lar concentration.

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1942 DEFRANK AND CHENG

A

o-

200 -

100-

o0

* BME (0.1)

* BME (1.0)

* DTT (0.1)

0 DTT (1.0)

6Incubation Time (Hours)

FIG. 7. Stimulatory effects of reducing agents on OPAA-2 hy-drolysis of DFP. BME, P-Mercaptoethanol.

the course of 6 months (1). To confirm this effect on OPAA-2activity by reducing agents, enzyme samples were incubatedat 4°C in assay medium with the addition of 0.1 or 1.0 mMDTT or ,3-mercaptoethanol. After 2, 6, and 18 h, the reactionmixtures were transferred to the assay vessel, brought up totemperature, and examined for activity by the addition ofDFP. Both reducing agents showed a stimulatory effect, withDTT giving the greatest increase (Fig. 7).

Effect of sulfhydryl inhibitors. The effects of several sulf-hydryl-specific inhibitors were examined. The inhibitorsp-chloromercuribenzoate (PCMB), iodoacetic acid (IAA),and N-ethylmaleimide (NEM) were added at 0.1, 1.0, and 5.0mM to the enzyme contained in the standard assay mediumand preincubated for 60 min at 4°C. The reaction mixtureswere then transferred to the assay vessel, warmed, andtested by the addition of DFP. All of these reagents causedan inhibition of OPAA-2 activity against DFP (Table 4). Theresults suggest that the enzyme requires a functional sulfhy-dryl group for its activity.

Effect of metals. The effects of various metal ions onOPAA-2 activity against DFP were examined in the follow-ing manner. The enzyme sample (as purified) was initiallytested under standard assay conditions, with the variousmetal ions added in place of manganese. The concentrationof metals used was set at 0.4 mM. A comparable sample ofenzyme, without any metal ion additions, was incubatedwith 0.1 mM EGTA [ethylenebis (oxyethylenenitrilo)tet-raacetic acid] for 60 min at room temperature and thenassayed for activity with DFP. Previous studies indicated

TABLE 4. Effects of sulfhydryl reagents on OPAA-2

% Activity'Reagent

0.1 mM 1.0 mM 5.0 mM

PCMB 92.4 80.3 69.5IAA 90.6 79.3 53.9NEM 60.5 48.7 34.9

a See Table 3, footnote b.

TABLE 5. Effects of metal ions on OPAA-2

% ActivityMetals tested Direct Post-EGTA 2nd MnCl2

addition addition addition

No addition 100.0a 4.6 124.0Type 1CoCl2 * 6H20 104.9 96.6 91.3MnCl2 4H20 124.0 104.6 104.6

Type 2CsCl 104.9 4.2 132.7CuC12 * 2H20 95.8 4.9 91.3FeCI3 * 6H20 102.7 10.6 99.2MgCl2 6H20 92.4 5.3 94.7NiCl2 * 6H20 121.7 12.2 103.0

Type 3CaCl2 *2H20 106.1 19.8 30.0ZnCl2 *7H20 31.2 8.4 8.7

a See Table 3, footnote b.

I8

that this time of incubation with EGTA would result in agreater than 90% inhibition of OPAA-2 but still permitcomplete recovery of activity upon the addition of manga-nese (2). To each EGTA-treated sample was added the metalion (at 0.4 mM) to be tested. A preincubation of 2 min wasconducted before the activity was measured. After the assayhas been completed, 0.4 mM MnCl2 was added to thereaction mixture to determine whether any additional stim-ulation could be obtained. The results of these assayssuggest that there are three groups of metal ion effects(summarized in Table 5).

DISCUSSION

Halophilic isolate JD6.5 possesses several OPA anhydraseactivities. Among all of the OPA anhydrases described fromvarious sources, the enzyme from JD6.5 has been shown tohave the highest activity. The predominant enzyme,OPAA-2, can be separated from other OPA anhydrases byDEAE-Sephacel chromatography. With DEAE-Sephacel atpH 5.0, OPAA-1 has been found to contain at least twoDFP-hydrolyzing activities (2).By use of the procedures described in this report, OPAA-2

has been purified to homogeneity with a purification factor ofgreater than 1,000-fold. SDS-PAGE indicates that the en-zyme is composed of a single polypeptide with a molecularweight of 60,000. This value corresponds well with theapparent molecular weight obtained in preliminary studiesusing Sephacryl S-200 gel permeation chromatography (2).Since several attempts to purify OPAA-2 by hydrophobicinteraction chromatography on phenyl-Sepharose CL-4Bwere unsuccessful because of the tight binding of the enzymeto the column, it can be inferred that it is a very hydrophobicprotein.The purified enzyme has been shown to possess high

activity against several organophosphorus compounds, in-cluding DFP, NPMPP, and NPEPP, with lesser activityagainst paraoxon. Preliminary nuclear magnetic resonanceexperiments (in the absence of manganese) indicated thatOPAA-2 hydrolyzes soman at 240% and sarin at 20% the rateof DFP, while tabun was not hydrolyzed at all (15). Allstereoisomers of soman and sarin were hydrolyzed at equalrates. Studies with the purified enzyme demonstrated thatOPAA-2 has a pH optimum of 8.5 and temperature optimumof 50°C. Under these conditions, the estimated turnover

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OPA ANHYDRASE FROM A HALOPHILE 1943

number for the enzyme, with DFP as a substrate, would beapproximately 100,000 molecules per min per enzyme mol-ecule. At room temperature, the turnover number is still arelatively high 35,000 min-'.The enzyme is reversibly inhibited by both EGTA and

Mipafox. Stimulation and stabilization of the enzyme areobserved with a reducing agent such as DTT or P-mercap-toethanol. The requirement for a sulfhydryl group wasdemonstrated by the inhibition of enzyme activity by PCMB,IAA, and NEM. This finding is also in line with the possiblepresence of a active sulfhydryl group (cysteine) suggested bythe pH studies. However, these results do not eliminate thepossibility of the involvement of a sulfhydryl group at alocation distant from the active site but important for thethree dimensional configuration of the enzyme or its stabil-ity.The inhibitory effect of EGTA was examined because it

binds divalent cations more strongly than does EDTA. At0.1 mM EGTA, a nearly complete inhibition of OPAA-2activity against DFP was observed. Similar attempts toinhibit the enzyme with EDTA showed only a slight effect(2). Three types of reactions were observed in regard to theeffects of metal ion additions to untreated or EGTA-treatedenzyme. In the first type, both manganese and cobaltshowed nearly complete reactivation of the enzyme activitywith no additional stimulation by further manganese addi-tion. The second group of metals (cesium, copper, iron,magnesium, and nickel) showed little or no reactivation ofthe enzyme, but they also did not block reactivation follow-ing manganese addition. The third group of metals (calciumand zinc) not only inhibited the enzyme to different degreesbut also prevented reactivation by manganese. Zinc, inparticular, showed significant inhibitory effect againstOPAA-2, both as an exogenous inhibitor and by blocking itsreactivation. In a number of instances, the reactivation ofthe enzyme by manganese following EGTA treatment re-sulted in much higher activity than that observed in theoriginal sample (2). These results suggest that as a result oflow levels of manganese or cobalt, a significant number ofenzyme molecules may have been loaded with either calciumor zinc, which would have rendered them inactive. Thepossible effect of the addition of manganese or other metalsto the growth media has yet to be investigated.

Polyclonal antiserum and monoclonal antibodies havebeen prepared against the purified OPAA-2. The character-ization of OPAA-2 by Western blotting analysis has beehcarried out with these antibodies. Additional preliminaryresults have shown that these antibodies react with proteinsin other halophilic and thermophilic bacteria that have beenpreviously shown to have DFP-hydrolyzing OPA anhy-drases (1). The results suggest that these functionally relatedenzymes may share common immunological determinantsbut do not eliminate the possibility of reactivity with com-pletely unrelated proteins.

Until relatively recently, it was thought that all of the OPAanhydrases were of two varieties that have been termedsquid type or Mazur type (6). In general, the squid-typeenzyme has a low molecular weight (under 40,000), isindifferent to manganese, Mipafox, and ammonium sulfate,and has a somanlDFP ratio of 0.2 to 0.3, with little or nostereoselectivity for soman isomers. Nearly all of otherenzymes fell into the Mazur-type category, with molecular

weight above 60,000, stimulation by manganese, inhibitionby Mipafox, lability in ammonium sulfate, and a soman/DFPratio greater than 1, with a preference for the nontoxicisomers of soman. This categorization of OPA anhydraseshas become much less clear as more enzymes are discoveredthat fall somewhere in between these two types (10). TheOPAA-2 from JD6.5 is a good example of this. It is Mazurtype in respect to its size (60,000), simulation or activationby manganese, inhibition by Mipafox, and soman/DFP ratio(approximately 2.4). However, it is squid like in its stabilityto ammonium sulfate and its lack of stereoselectivity withsoman. Clearly, classification of the OPA anhydrases willrequire a great deal more information on the physical andbiochemical parameters of the ever-increasing number ofthese enzymes. At this point, it is still impossible to specu-late on what the natural substrate of OPAA-2 might be orwhat role it plays in the normal metabolism of JD6.5.

ACKNOWLEDGMENTS

We are grateful to Sammy Liu, Johns Hopkins University, andMaryalice Miller, CRDEC, for the preparation of rat polyclonalantiserum and monoclonal antibody, respectively.

REFERENCES1. Cheng, T.-c., and J. J. DeFrank. Unpublished data.2. DeFrank, J. J. Unpublished data.3. DeFrank, J. J., and T.-c. Cheng. 1989. Characterization of a

diisopropylfluorophosphate hydrolyzing enzyme from an obli-gate halophile, abstr. K-186, p. 276. Abstr. 89th Annu. Meet.Am. Soc. Microbiol. 1989.

4. Fersht, A. 1985. Enzyme structure and mechanism. W. H.Freeman and Co., New York.

5. Hoskin, F. C. G. 1985. Inhibition of a soman- and diisopropylphosphorofluoridate (DFP)-detoxifying enzyme by mipafox.Biochem. Pharmacol. 34:2069-2072.

6. Hoskin, F. C. G., M. A. Kirkish, and K. E. Steinmann. 1984.Two enzymes for the detoxification of organophosphorus com-pounds-sources, similarities, and significance. Fund. Appl.Toxicol. 4:165-172.

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8. Kennet, R. H. 1980. Fusion by centrifugation of cells suspendedin polyethylene glycol, p. 365-367. In R. H. Kennet, T. J.McKeam, and K. B. Bechtol (ed.), Monoclonal antibodies.Plenum, New York.

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