Purification and Crystallization of the Oxygenase Component of Naphthalene Dioxygenase in Native and Selenomethionine-Derivatized Forms

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 241, 553–557 (1997)ARTICLE NO. RC977863

Purification and Crystallization of the OxygenaseComponent of Naphthalene Dioxygenase in Nativeand Selenomethionine-Derivatized Forms

Kyoung Lee,*,1 Bjorn Kauppi,† Rebecca E. Parales,* David T. Gibson,*,2 and S. Ramaswamy†*Department of Microbiology and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa52242; and †Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden

Received October 21, 1997

oxidizes naphthalene to (/)-(1R,2S)-dihydroxy-1,2-di-A new procedure was developed for the purification hydronaphthalene (3). In this reaction electrons are

of the terminal oxygenase component (ISPNAP) of naph- transferred through an iron-sulfur flavoprotein (Reduc-thalene dioxygenase. From a five liter culture of Esche- taseNAP (4)) to a Rieske [2Fe-2S] protein (FerredoxinNAPrichia coli JM109(DE3)(pDTG121), 91 mg of pure pro- (5)). The latter reduces the terminal oxygenase whichtein were obtained with a specific activity of 2.48 mmol/ is an iron-sulfur protein (ISPNAP (6)). ISPNAP has twomin/mg protein. ISPNAP was crystallized in the rhombo-

different subunits (ab). Each a subunit (55-kDa) con-hedral space group R32 with cell dimensions oftains a Rieske [2Fe-2S] center and non-heme mononu-aÅbÅ179.2 A; cÅ322.5A in the hexagonal setting. Theclear iron (6-8). The nucleotide sequences of all fourcrystals are brown, indicating the presence of an in-structural genes have been determined (9, 10) and aretact Rieske iron-sulfur center. Problems with non-iso-available through the GenBank database (accessionmorphism between native data sets necessitated thenumbers M83950 and U49496).preparation of a selenomethionine-substituted pro-

Substrate specificity studies have shown that NDOtein. Complete replacement of methionine with seleno-can catalyze enantiospecific cis-dihydroxylation, mono-methionine was achieved and the purified protein hadhydroxylation and sulfoxidation reactions. In addition,a specific activity almost identical to native ISPNAP.

Crystals from this preparation belong to the same the enzyme catalyzes desaturation, alcohol oxidation,space group and have similar cell dimensions to native N- and O-dealkylation reactions (for a review, see ref.ISPNAP. q 1997 Academic Press 11). These observations show that NDO can play a role

in areas as diverse as bioremediation and the synthesisof biologically active compounds (12).

To date, more than 30 multicomponent non-heme ironAromatic hydrocarbons are common environmentaloxygenases containing Rieske [2Fe-2S] centers havepollutants. Interest in this class of compounds stemsbeen identified by sequence analysis or protein purifica-from the fact that many of them are toxic and in sometion. We now report the purification, crystallization andcases cause cancer in experimental animals. The aero-preliminary X-ray analysis of the terminal oxygenasebic degradation of many aromatic hydrocarbons by bac-component (ISPNAP) of NDO. To our knowledge this isteria is initiated by dioxygenases, which catalyze thethe first report of the crystallization of an oxygenaseenantiospecific addition of dioxygen to the aromaticcomponent from this unique group of enzymes.ring to form cis-dihydrodiols. Substrates ranging in

size from benzene to benzo[a]pyrene are known to beMATERIALS AND METHODSoxidized through these intermediates (1). A representa-

tive example of this type of enzyme is naphthalene di-Bacterial strains and growth conditions. Escherichia colioxygenase (NDO) which is induced in Pseudomonas sp. JM109(DE3)(pDTG121) (13), which contains the cloned nahAcAd

strain NCIB 9816-4 during growth with naphthalene genes encoding the a and b subunits of ISPNAP, was grown and in-(2). NDO is a three component enzyme system that duced with isopropyl-b-D-thiogalactopyranoside (IPTG) as described

previously (14). E. coli GS765(pDTG165), a methionine auxotroph(15) that expresses ISPNAP, was constructed as follows. From theplasmid pDTG141 (16), a 1.7-kb XhoI-HindIII fragment carrying the1 Present address: Department of Microbiology, Changwon Na-

tional University, Changwon-Si, Kyongnam, South Korea. nahAcAd genes was ligated into pTrc99A (Pharmacia Biotech. Inc.,Piscataway, NJ) that had been digested with SalI and HindIII. The2 Corresponding author: Fax: (319) 335-9999. E-mail: david-gibson@

uiowa.edu. recombinant strain GS765(pDTG165), which contains the nahAcAd

0006-291X/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

553

AID BBRC 7863 / 6942$$1301 11-25-97 15:07:26 bbrcg AP: BBRC

Vol. 241, No. 2, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. The X-ray absorption spectrum of a SeMet-substituted ISPNAP crystal, when scanning the energy interval 12.6107 to 127502 kEV.

genes under the control of an IPTG-inducible trc promoter, was used JM109(DE3)(pDTG121) was applied to a Q-Sepharose FF column (5for the purification of selenomethionine-substituted ISPNAP. The re- 1 20 cm) previously equilibrated with BTGD buffer. The column wascombinant strain was grown in 5 liters of mineral salts medium washed with 400 ml of BTGD buffer containing 0.1 M KCl at a flow(MSB) (17) containing glucose (4 g/l), L-methionine (50 mg/l), thia- rate of 2.0 ml/min. Bound ISPNAP was eluted with a 800-ml linearmine (8 mg/l) and ampicillin (200 mg/l), in a New Brunswick Scien- KCl gradient (0.1-0.5 M) in BTGD buffer. Fractions (18 ml) weretific BiofloII 6 l fermentor. The culture was maintained at 277C with collected and assayed for NDO activity. Fractions (41-50) werestirring (300 rpm) and aeration (3 vvm). When the turbidity at 660 pooled, desalted and concentrated to 11.7 ml (655 mg of protein) overnm reached 2.5, the cells were harvested by centrifugation to yield an Aminco YM100 membrane by ultrafiltration. Membrane-filtered51 g cells (wet weight). Two thirds of the harvested cells were washed 4.0 M (NH4)2SO4 was added to give a final concentration of 1.0 Mtwice with MSB medium, resuspended to a turbidity of 1.5 at 660 (NH4)2SO4. After removing the precipitate by centrifugation, the su-nm in MSB medium containing glucose, thiamine and ampicillin, pernatant was applied to a column (2.61 27.5 cm) of Octyl Sepharoseand cultured in the same fermentor at 277C with stirring (400 rpm) CL-4B equilibrated with BTGD buffer containing 1.0 M (NH4)2SO4.and aeration (3 vvm) for 2 h to deplete intracellular methionine. At ISPNAP was eluted with a 300 ml linear gradient of decreasingthis time IPTG (300 mg), DL-selenomethionine (SeMet, 200 mg), and (NH4)2SO4 (1.0-0.2 M) in BTGD buffer followed by isocratic elutionFeCl3r6H2O (0.5 g) were added to the culture. Cells were harvested with 300 ml of BTGD buffer containing 0.2 M (NH4)2SO4. Fractionsby centrifugation after 2.5 h when the turbidity at 660 nm was 3.0, (4 ml) were collected. Active fractions (102-128) were pooled, concen-and stored at 0707C. trated to 6.4 ml by ultrafiltration, and reequilibrated with BTGD

containing 0.1 M KCl by ultrafiltration. The concentrated samplePurification of ISPNAP and enzyme assay. Crude cell extracts were(154 mg of protein) was applied to a column (2.6 1 100 cm) of Sepha-prepared as described previously (18). The buffer used for breakagecryl S-300 equilibrated with BTGD containing 0.1 M KCl. ISPNAPwas BTGD buffer (50 mM Bis-Tris [pH6.8], 5% glycerol, 1 mM sodiumwas eluted at a flow rate of 0.5 ml/min and fractions (2 ml) weredithiothreitol) containing 0.1 mM phenylmethylsulfonyl fluoride andcollected. Active fractions (140-155) were pooled and washed with1 mg/ml of DNase. Protein purification was conducted at 47C with aBTGD by ultrafiltration. Purified ISPNAP was drop-frozen in liquidFPLC system (Pharmacia Biotech, Piscataway, N.J.). A crude cell

extract (4.1 g of protein) obtained from 54 g of IPTG-induced nitrogen in 25 ml aliquots and the protein pellets were stored at

554



FIG. 2. The crystals of ISPNAP grew as hexagonal plates in three days to a size of 0.5 1 0.5 1 0.1 mm3. They belong to the space groupR32 and are brown in color indicating an intact Rieske iron-sulfur center.

0707C. SeMet-substituted ISPNAP was purified according to the pro- RESULTS AND DISCUSSIONtocol established for native ISPNAP. The activity was measured po-larographically with an oxygen electrode as described previously (19).

Although active ISPNAP has been purified previouslyGel electrophoresis and Western blot analysis. Protein purity wasdetermined by electrophoresis in sodium dodecyl sulfate polyacryl- (6, 13), further optimization of the purification condi-amide gels (SDS-PAGE, 12%) as described by Laemmli (20). Protein tions were required to obtain large amounts of proteinbands were stained with Coomassie blue. Western blots with poly- for crystallization. The procedure described in Materi-clonal antibody raised against ISPNAP were carried out as previously

als and Methods gave 91 mg of ISPNAP, with a specificdescribed (21).activity of 2.48 mmol/min/mg ISPNAP. The preparationCrystallization. Initial crystallization experiments were con-from 5 liters of medium was homogeneous when ana-ducted with Crystal Screens I and II supplied by Hampton Research,

Riverside, CA. Specific details of the procedure are given in the Re- lyzed by SDS-PAGE and gave the same absorptionsults and Discussion section. spectrum as reported previously (6, 13).

X-ray diffraction experiments. One crystal was mounted in a ny- Growth of GS765(pDTG165) was inhibited in thelon loop and frozen in a flash nitrogen stream at 1007K using 20- presence of SeMet. To circumvent this problem, cells25% ethylene glycol as a cryo-protectant while maintaining the ionic

were grown to a high cell density in the presence ofstrength of the precipitate. The exact amount of ethylene glycol usedmethionine. They were then starved for methioninewas dependent on the humidity. Data was initially collected on a

rotating anode with an RAXIS-II imaging plate detector. Space group prior to IPTG induction of the NDO genes in the pres-and cell dimensions were measured by auto indexing 17 oscillation ence of SeMet. Western blot analysis with polyclonalpictures using Denzo (22). ScalePack (22) was used to merge and antibody raised against purified ISPNAP showed thatscale all data together. Native data were collected at synchrotron

cells, prior to induction, contained barely detectablebeam lines BW7B at EMBL/DESY, Hamburg and at ID2 at ESRF,levels of ISPNAP. The same purification procedure de-Grenoble using a MAR-imaging plate detector. An EXAFS scan of

a selenium-containing ISPNAP crystal was measured by aligning a scribed above gave 12.3 mg of homogeneous SeMet-fluorescent sensitive detector vertical to the incoming beam while substituted ISPNAP with a specific activity of 2.19 mmol/scanning over the energy interval 12.6107 - 12.7502 keV at BM14, min/mg SeMet-substituted ISPNAP from a 5 liter cul-ESRF, Grenoble. Later, a multi wavelength anomalous diffraction

ture. Electrospray/mass spectrometry of native ISPNAP(MAD) data set were measured at the identified K-absorption edgeof selenium at the same station. gave a molecular mass of 49,613 { 10-Da for the a

555



subunit. Two peaks at 22,805- and 22,932 { 4-Da were peak was found at approximately 0,0,1/2. This indi-cates a pseudo symmetry translation. Native data setsobtained for the b subunit. The deduced molecular

masses of the a and b subunits of ISPNAP are 49,612-Da of the R32 form collected from different crystals provedto be non-isomorphous. For example, the two best na-and 22,937-Da, respectively. In addition, the deduced

amino acid sequences show that the a and b subunits tive data sets have a Riso (on F’s) of 23.1% when scaledin Scaleit (25). This problem can be eliminated by usingcontain 10 and 5 methionine residues, respectively. The

partial loss of the N-terminal methionine in the b sub- pure MAD techniques as all data sets can be collectedfrom a single frozen crystal (26). Overall, the R32 crys-unit accounts for the two values obtained and presum-

ably occurs during processing by E. coli. The experi- tals exhibit the appropriate attributes for further in-vestigation, and the determination of the structure ofmentally determined mass of the a subunit of SeMet-

substituted ISPNAP was 50,081-Da and masses of the ISPNAP is in progress.b subunit were 23,169- and 22,992-Da. These resultsconfirm the complete replacement of methionine with ACKNOWLEDGMENTSSeMet in SeMet-substituted ISPNAP. These include 10SeMet residues in the a subunit and 5 or 4 residues in This work was supported by US Public Health Service Grantthe b subunit. The EXAFS spectra show a distinct peak GM29909 from the National Institute of General Medical Sciences (to

D.T.G.) and by the Swedish Natural Research Council and Swedisharound the expected energy for selenium X-ray absorp-Research Council for Agricultural and Forestry (to Hans Eklund).tion (Fig. 1).We thank Arthur Arnone, Paul H. Rogers and Sang-Kee Rhee forThe native enzyme was crystallized using the vapor helpful discussions during the early stages of crystallization, George

equilibrium procedure. The ISPNAP solution (40-50 mg V. Stauffer and Mark Urbanowski for E. coli GS765, and Andyof protein/ml) was mixed with an equal volume of reser- Thompson for help with EXAFS measurements and data collection

on BM14, ESRF, Grenoble. The electrospray mass spectra were pro-voir solution on a siliconized microscope cover slip. Thevided by Donald H. Seielstad at the Bioanalytical Services Facilitycover slip was inverted and sealed over a microtiterat the University of Illinois. We also thank the Swedish Naturalwell containing 0.3 ml of reservoir solution. The first Research Council for travel grants to the synchrotron at EMBL/

crystal form was found in 2.0 M (NH4)2SO4, 100 mM DESY, Hamburg. Last, we thank Bryce V. Plapp for providing theMES buffer (pH 5.5) and 2.5% MPD at 87C. The crystals essential link between our two research groups.belong to class P422 with aÅbÅ176.4A, cÅ536.3A anddiffracted to 2.9 A when a few degrees of data were REFERENCESevaluated on beam line X12B at National SynchrotronLight Source, Brookhaven National Laboratory. The 1. Gibson, D. T., and Subramanian, V. (1984) in Microbial Degrada-very long c-axis was not found using a rotating anode tion of Organic Compounds (Gibson, D. T., Ed.), pp. 181–251,

Dekker, New York.and a synchrotron radiation x-ray source was required2. Ensley, B. D., Gibson, D. T., and Laborde, A. L. (1882) J. Bacte-to separate the spots and thus discover the long axis.

riol. 149, 948–954.The absolute requirement for synchrotron radiation3. Jeffrey, A. M., Yeh, H. J. C., Jerina, D. M., Patel, T. R., Davey,make these crystals less useful for de novo structure

J. F., and Gibson, D. T. (1975) Biochemistry 14, 575–583.determination. However, a different crystal form grew4. Haigler, B. E., and Gibson, D. T. (1990) J. Bacteriol. 172, 457–in a solution which contained 0.3-0.65 M sodium sul-

464.fate, 100 mM MES buffer (pH 5.5), 5.0 mM NiSO4, and5. Haigler, B. E., and Gibson, D. T. (1990) J. Bacteriol. 172, 465–200 mM 1,6-hexanediol. Crystals grew to a full size of 468.

0.5 1 0.5 1 0.1 mm3 after 3 days at 47C. SeMet-substi- 6. Ensley, B. D., and Gibson, D. T. (1983) J. Bacteriol. 155, 505–tuted ISPNAP crystals were obtained when the pH was 511.raised to 6.0 and the temperature to 87C. Crystal for- 7. Suen, W.-C., and Gibson, D. T. (1993) J. Bacteriol. 175, 5877–mation was not observed in the absence of Ni2/ ions or 5881.at protein concentrations of less than 20 mg/ml. Both 8. Jiang, H., Parales, R. E., Lynch, N. A., and Gibson, D. T. (1996)

J. Bacteriol. 178, 3133–3139.crystal types are thin, whole or half hexagonal plateswhich are brown indicating an intact Rieske iron-sulfur 9. Simon, M. J., Osslund, T. D., Saunders, R., Ensley, B. D., Suggs,

S., Harcourt, A., Suen, W.-C., Cruden, D. L., Gibson, D. T., andcenter (Fig. 2). They belong to the rhombohedral spaceZylstra, G. J. (1993) Gene 127, 31–37.group R32 with cell dimensions aÅbÅcÅ149.1A and

10. Parales, J. V., Kumar, A., Parales, R. E., and Gibson, D. T. (1996)aÅbÅgÅ74.37, corresponding to aÅbÅ179.2A,Gene 181, 57–61.cÅ322.5A in the hexagonal setting. The crystals dif-

11. Resnick, S. M., Lee, K., and Gibson, D. T. (1996) J. Indust. Micro-fract to approximately 3 A on an in-house rotating biol. 17, 438–457.anode and to 2.5 A on a synchrotron. The asymmetric

12. Hudlicky, T., and Reed, J. W. (1995) in Advances in Asymmetricunit is occupied by two molecules (a2b2) resulting in a Synthesis (Hassner, A., Ed.), Vol. 1, pp. 271–312, JAI Press,calculated solvent content of 60% and a Vm Å 3.47 A3/ Greenwich, CT.Da (23). No peaks were detected when a self rotation 13. Suen, W.-C., and Gibson, D. T. (1994) Gene 143, 67–71.plot was calculated in GLRF (24). However, in a native 14. Lee, K., Resnick, S. M., and Gibson, D. T. (1997) Appl. Environ.

Microbiol. 63, 2067–2070.Patterson map a huge peak about 50 % of the origin

556



15. Mares, R., Urbanowski, M. L., and Stauffer, G. V. (1992) J. Bac- 21. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seid-man, J. G., Smith, J. A., and Struhl, K. (1993) Current Protocolsteriol. 174, 390–397.in Molecular Biology, Wiley, New York.16. Suen, W.-C. (1991) Ph.D. thesis. University of Iowa, Iowa City.

22. Otwinowski, Z. (1993) Proceedings of the CCP4 Study Weekend,17. Stanier, R. Y., Palleroni, N. J., and Doudoroff, M. (1966) J. Gen. Warrington, UK.

Microbiol. 43, 159–271.23. Matthews, B. W. (1968) J. Mol. Biol. 33, 491–497.

18. Haddock, J. D., Nadim, L. M., and Gibson, D. T. (1993) J. Bacte- 24. Tong, L., and Rossman, M. G. (1990) Acta Crystallog. Sect. A 46,riol. 175, 395–400. 783–792.

19. Lee, K., and Gibson, D. T. (1996) Appl. Environ. Microbiol. 62, 25. The CCP4 suite: Programs for protein crystallography. (1994)3101–3106. Acta Crystallog. Sect. D 50, 760–763.

26. Hendrickson, W. A. (1991) Science 254, 51–58.20. Laemmli, U. K. (1970) Nature (London) 227, 680–685.

557


Documents

Purification and Crystallization of the Oxygenase Component of Naphthalene Dioxygenase in Native and Selenomethionine-Derivatized Forms