PROTEINS Structure, Function, and Genetics 11233-236 (1991)

Crystallization and Preliminary X-Ray Investigation of a Recombinant Form of Rat Catechol 0-Methyltransferase Jukka Vidgren,'s2 Carola Tilgmann,',3Kenneth Lundstrom,' and Anders Liljas2 'Orion Pharmaceutica, Research Center, SF-021 01 Espoo, Finland, 2Department of Molecular Biophysics, Chemical Center, University of Lund, S-22100 Lund, Sweden. and 3Znstitute of Biotechnology, University of Helsinki, SF-00380 Helsinki, Finland

ABSTRACT Rat catechol 0-methyltransferase cDNA was introduced into an E. coli expression vector pKEX14, which utilizes the inducible T7 promoter. Active and soluble recombinant catechol 0-methyltransferase was produced in bacteria and purified to electrophoretic homogeneity by chromatographic procedures. The purified enzyme has been crystallized by the method of vapor diffusion using polyethylene glycol as precipitant. The space group is P3,21 or P3,21 with a = b = 51.3 A and c = 168.5 A and one molecule in the asymmetric unit. The crystals diffract beyond 3.2 A and are suitable for three-dimensional X-ray structure determination. Key words: protein crystals, X-ray crystallography, catechol 0-methyltransferase, protein structure INTRODUCTION Catecholo-methyltransferase(COMT,S-adenosylL-methionine: catechol 0-methyltransferase, EC 2.1.1.6) catalyzes the transfer of the active methyl group from S-adenosyl-L-methionine to one of the two ring hydroxyl groups of catechols.' The COMT enzyme is widely distributed in mammalian tissues. Substrates of COMT include both endogenous catechols, such as catecholamine neurotransmitters and catechol steroids as various catechol-type xenobioti~s.',~ This enzyme also plays an important role in inactivation of many neuroactive drugs such as L-Dopa, a-methyldopa, and is~proterenol.~ The presence of a divalent cation such as Mgz+ is essential for the enzymatic a ~ t i v i t y . ' , ~The , ~ favorable effect of the COMT inhibition in therapy of Parkinson's disease has been r e p ~ r t e d ,leading ~ . ~ to the development of synthetic COMT inhibitom8 To study the interaction of the COMT enzyme and inhibitors, rat liver cDNA has been cloned.' The amino acid sequence of COMT consisting of 221 amino acid residues (M, = 24,747) was determined from the nucleotide sequence and compared to that obtained from peptide sequencing of highly purified rat liver C0MT.I' The purification of COMT enzyme 0 1991 WILEY-LISS, INC.

from rat tissues yielded too low amounts to allow structural characterizations of the protein. However, the expression of the cloned rat liver COMT in Escherichia coli resulted in sufficient amounts of recombinant COMT to be purified and used for crystallization experiments. In this paper we report the crystallization and a preliminary crystallographic study of the recombinant form of rat liver COMT.

MATERIALS AND METHODS Expression and Purification of the Catechol 0-Methyltransferase The cDNA of soluble COMT was cloned from the rat liver cDNA library.' The coding region of 663 nucleotides was introduced into a bacterial expression vector pKEX14, which is a modification of the $EX2 vector." The upstream ATG initiation codon a t the NcoI site from vector pJEX2 has been deleted by mung bean nuclease treatment before inserting the PCR-generated COMT coding region into the EcoRI-Hind111 sites of the pKEX14 vector. Soluble catechol 0-methyltransferase was produced in E. coli by first growing the bacteria at 37°C to midlogarithmic phase in 4 liter yeast tryptone containing 100 ygiml ampicillin. Production of soluble catechol 0-methyltransferase was then induced with isopropyl-P-D-thiogalactopyranoside,and the cells harvested after an additional growth of 3 hr. Harvested cells (16 g wet weight) were disrupted by sonication a t 4°C in 160 ml sodium phosphate buffer, pH 7.0. The obtained mixture was clarified by centrifugation for 30 min at 10,OOOg (at 4°C) and the supernatant concentrated to 60 ml by ultrafiltration (Filtron Novacell NC 10, 150 ml). Gel filtration was performed on a BioGel P-100 column (5.0 x 90 cm)

Accepted for publication April 29, 1991. Address reprint requests to Jukka Vidgren, Orion Pharmaceutica, Research Center, Box 65, SF-02101 Espoo, Finland. Abbreviations: COMT, catechol 0-methyltransferase; AdoMet, S-adenosyl-L-methionine; MES, morpholineethane 4sulfonic acid PEG 6000, polyethylene glycol with average molecular weight of 6000; V,, the ratio of unit cell volume to molecular weight (A3/Da); SDS, sodium dodecyl sulfate; SDSPAGE, SDS polyacrylamide gel electrophoresis.

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A.

8. a 94 k

b

68

b

43

b

30

b

b

20,l b 14,4

F

0

10

20

30

40 min.

Fig. 1. Purity control of the recombinant rat-liver COMT used for crystallization.(A) Reversed-phase chromatography on a TSK TMS250 (0.46 x 3 cm) column. Of the recombinant COMT 10 pg was loaded on the column and chromatography performed with a linear gradient of acetonitrile (0-100% B in 60 min) in 0.1% tri-

fluoroacetic acid. (B) SDS-PAGE of (a) a standard mixture containing proteins with molecular masses of 94, 68, 43, 20.1, 14.4 kDa; (b) 2.5 p,g of recombinant COMT. Electrophoresis was performed in a 12.5% polyacrylamide gel and the proteins stained with Coomassie Brilliant Blue.

equilibrated with 20 mM bis-Tris-HC1, pH 5.8, and the collected fractions were subjected to COMT activity measurements as described COMT-active fractions were pooled and subjected to anion exchange chromatography on a Q-sepharose HL 16/10 column (Pharmacia) equilibrated with the gel filtration buffer. Elution was performed with a linear gradient of sodium chloride (0-0.5 M) in the equilibration buffer. The COMT-active fractions were pooled and concentrated by ultrafiltration as above, and the buffer changed to 20 mM bis-TrisHC1, pH 5.8, 10 mM dithiothreitol in a BioGel P6DG column. The COMT-active fraction from this column was applied to a Mono-Q HR 5/5 column (Pharmacia) equilibrated with the previous buffer and chromatography performed with a linear gradient of sodium chloride (0-0.5 M) in the equilibration

buffer. The COMT-active peak was collected and used for the crystallization experiments. The purity of the collected active fraction was controlled by SDS polyacrylamide gel electroph~resis'~ and reversedphase-high-performance liquid chromatography.

Crystallization of the Catechol 0-Methyltransferase For crystallization experiments of COMT the sample was concentrated to 4.5 mg/ml in 50 mM MES buffer solution containing 2.5 mM MgCl,, 1.5 mM AdoMet, 0.7 mM 1,5-dinitrocatechol, and 0.5 mM dithiothreitol at pH 6.5. Crystals suitable for X-ray structural analysis were grown by the method of vapor diffusion in hanging drops. Each hanging drop contained 5.0 p1 of protein solution, and 5.0 p1 of 8% (wiv) PEG 6000 dissolved in 50 mM MES buffer at

CRYSTAL DATA FOR CATECHOL 0-METHYLTRANSFERASE

235

pH 6.5. The hanging drops were equilibrated a t 22°C against a reservoir solution containing 10 ml of 0.5 M sodium chloride. After 4-5 days at room temperature, hexagonal-shaped crystals with dimensions up to 0.30 x 0.25 x 0.20 mm grew from these droplets. Crystals were mounted in thin-walled glass capillaries measuring 0.7 mm in diameter.

X-Ray Diffraction Analysis X-Ray diffraction data were collected using a Siemens multiwire electronic area detector. CuK, X-ray radiation was generated using a Rigaku RU2OOHB rotating anode X-ray generator with a 0.3 x 3 mm focus running at 120 mA and 35 kV. The rotating anode was equipped with a graphite monochromator and the beam was collimated to 0.5 mm. The area detector was mounted 13 cm from the crystal with a fixed x = 45", 2 0 = 0" intercepting data from 50 t o 3.8 A. Diffraction data was collected as a series of discrete frames or electronic images each comprising 0.3" oscillation of o-axis. The exposure time per frame was 150 sec. The raw data frames were transferred to a VAXstation 3100 for subsequent processing. The preliminary crystallographic analysis was done using only the electronic areadetector data; no X-ray precession photographs were taken due to the weak diffraction. The determination of unit cell parameters, crystal orientation, and integration of reflection intensities was performed with the XENGEN program suite.'* RESULTS AND DISCUSSION The coding region of rat liver COMT expressed from the T7 promotor in vector pKEX14 yielded approximately 15% recombinant COMT of total protein in E . coli. This active and soluble protein was purified as described in Materials and Methods. High purity, up to 98%, was obtained (Fig. 1).Crystals were obtained from a mixture of COMT, AdoMet, and inhibitor. The crystals appeared after about 4 days of incubation a t 22°C and grew to full size within 7-10 days obtaining maximum dimensions of 0.3 mm (Fig. 2). From the attempts to crystallize COMT without the cosubstrate AdoMet and the inhibitor 1,Bdinitrocatechol only needle-like crystals resulted, which were not suitable for further crystallographic investigations. Crystals from the hanging drop were collected, dissolved, and subjected to a SDS polyacryl amide gel electrophoretic analysis. The mobility of the redissolved crystal and the purified starting material was identical (data not shown). Diffraction data from three single COMT crystals were used to determine unit cell dimensions and the space group. Area detector frames with exposure times of 150 sec and a rotation range of 0.3" show diffraction to 3.2 A resolution (Fig. 3). The X-ray data collected from a single crystal included 11,165 observations of 2,541 reflections of 2,706 possible ones at 3.8 A resolution. The data

Fig. 2. Crystal of recombinant rat catechol Ornethyltransferase grown using the hanging-drop vapor diffusion procedure. Crystals were obtained at 22°C from droplets consisting of 5 kl of 4.5 rng/rnl protein solution containing 2.5 rnM MgCI,, 1.5 rnM AdoMet, 0.7 rnM 1,5-dinitrocatechoI,and 0.5 rnM dithiothreitol in 50 rnM Mes and 5 kI of 8% PEG 6000 at pH 6.5. The size of the shown crystal is 0.3 x 0.25 x 0.25 rnrn.

scaled with symmetry R factors on intensities of R, = 9.5 and R,, = 9.3 and an average I/a(n of 9.7. The unit cell is trigonal with dimensions a = b = 51.3 and c = 168.5 A. The space group is P3,21 or P3,21 based on systematic absences of reflections along the 001 axis and the Laue symmetry (3m), was determined using the integrated diffraction data and the program XPREP.15 The (0,0,1) reflections with 1 = 3 n had Z/a(O > 10; the intensities of the reflections 1 3n had ZlaQ < 2. Assuming one molecule in the asymmetric unit with M, = 24,747, the V, is 2.5 A3/Da. This value is close to the average (2.45 A3/Da) for crystals of proteins of this molecular weight as observed by Matthews." The search for heavy atom derivatives is in progress.

ACKNOWLEDGMENTS This work was supported by Nordic Industrial Fund (J.V.), which is gratefully acknowledged. We are grateful for equipment funds from the SE-bank, the Knut and Alice Wallenberg foundation, and the

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Fig. 3. X-Ray diffraction pattern (0.3” oscillation) of a catechol Omethyltransferase crystal, recorded on a Siemens area detector (D = 13 cm, 20 = 5”). The maximum resolution in the picture is 3.2 8,

Swedish Council for Planning and Coordination of Research (FRN).

REFERENCES 1. Axelrod, J.,Tomchick, R. Enzymatic 0-methylation of epinephrine and other catechols. J. Biol. Chem. 233:702-705, 1958. 2. Guldberg, H., Marsden, C., Catechol-0-methyl transferase: Pharmacological aspects and physiological role. Pharmacol. Rev. 27:135-206, 1975. 3. Ball, P., Knuppen, R., Haupt, M. Breuer, H. Interactions between estrogens and catechol amines 111. Studies on the methylation of catechol estrogens, catechol amines and other catechols by the catechol-0-methyltransferaseof human liver. J. Clin. Endocrinol. 34:736-746, 1972. 4. Senoh, S., Tokuyama, Y., Witkop, B. The role of cations in non-enzymatic and enzymatic 0-methylations of catechol derivatives. J. Am. Chem. SOC.841719-1724, 1962. 5. Jeffery, D., Roth, J. Kinetic reaction mechanism for magnesium binding to membrane-bound and soluble catechol 0-methyltransferase. Biochemistry 26:2955-2958, 1987. 6. Linden, I-B, Nissinen, E., Etemadzadeh, E., Kaakkola, S., Mannisto, P., Pohto, P. Favorable effect of catechol-0methyltransferase inhibition by OR-462 in experimental models of Parkinson’s disease. Pharmacol. Exp. Ther. 247: 289-293, 1988. 7. Mannisto, P., Kaakkola, S. New selective COMT inhibitors: Useful adjuncts for Parkinson’s disease?. Trends Physiol. Sci. 1054-56, 1989.

8. Backstrom, R., Honkanen, E., Pippuri, A,, Kairisalo, P., Pystynen, J., Heinola, K., Nissinen, E., Linden, I-B., MBnnisto, P., Kaakkola, S., Pohto, P. Synthesis of some novel potent and selective catechol 0-methyltransferase inhibitors. J. Med. Chem. 32:841-846, 1989. 9. Salminen, M., Lundstrom, K., Tilgmann, C., Savolainen, R., Kalkkinen, N., Ulmanen, I. Molecular cloning and characterization of rat liver catechol-0-methyltransferase. Gene 93241-247,1990. 10. Tilgmann, C., Kalkkinen, N. Purification and partial characterization of rat liver soluble catechol-0-methyltransferase. FEBS Lett. 26495-99, 1990. 11. Peranen, J. Ph.D. Thesis, University of Helsinki, 1990. 12. Schultz, E., Nissinen, E. Determination of catechol-omethyltransferase activity in erythrocytes by high performance liquid chromatography with electrochemical detection. Biomed. Chromatogr. 3:64-67,1989. 13. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680-685, 1970. 14. Howard, A.J., Gilliland, G.L., Finzel, B.C., Poulos, T.L., Ohlendorf, D.H., Salemme, F.R. The use of an imaging proportional counter in macromolecular crystallography. J. Appl. Crystallogr. 20:383-387, 1987. 15. XPREP SHELXTL-PLUS Reciprocal Space Plotting Program Siemens Analytical X-ray Insts. Inc., 1990. 16. Matthews, B.W. X-Ray structure of proteins. In: “The Proteins” (Neurath, H., Hill, R.H., eds.), Vol. 111. New York Academic Press. 1977: 403-590.

Crystallization and preliminary X-ray investigation of a recombinant form of rat catechol O-methyltransferase.

Rat catechol O-methyltransferase cDNA was introduced into an E. coli expression vector pKEX14, which utilizes the inducible T7 promoter. Active and so...
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