Crystals of the Saccharomyces cerevisiae pyrimidine biosynthetic enzyme orotidine 5'-phosphate decarboxylase (ODCase) were grown by the hanging-drop vapor-diffusion technique at 277 K using polyethylene glycol 4000 as the precipitant. Crystals of native and selenomethionyl ODCase diffract to less than 2.2 A and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 90.1, b = 116.2, c = 117.0 A. Crystals of ODCase grown in the presence of the postulated transition-state analog inhibitor 6-hydroxyuridine 5'--phosphate (BMP) diffract to less than 2.5 A and belong to space group P2(1), with unit-cell parameters a = 79.9, b = 80.0, c = 98.2 A, beta = 108.6 degrees

Hassell, Anne

Milburn, Michael

Miller, Brian

Short, Steven

English

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472 Miller et al.  Orotidine 50-phosphate decarboxylase Acta Cryst. (2000). D56, 472±474crystallization papersActa Crystallographica Section DBiologicalCrystallographyISSN 0907-4449Crystallization of native and selenomethionyl yeastorotidine 50-phosphate decarboxylaseBrian G. Miller,a Anne M.Hassell,b Michael V. Milburnband Steven A. Shortc*aDepartment of Biochemistry and Biophysics,University of North Carolina, Chapel Hill,NC 27599-7260, USA, bStructural ChemistryDepartment, GlaxoWellcome, 5 Moore Drive,Research Triangle Park, NC 27709, USA, andcMolecular Sciences Department,GlaxoWellcome, 5 Moore Drive, ResearchTriangle Park, NC 27709, USACorrespondence e-mail:sas44336@glaxowellcome.com# 2000 International Union of CrystallographyPrinted in Denmark ± all rights reservedCrystals of the Saccharomyces cerevisiae pyrimidine biosyntheticenzyme orotidine 50-phosphate decarboxylase (ODCase) were grownby the hanging-drop vapor-diffusion technique at 277 K usingpolyethylene glycol 4000 as the precipitant. Crystals of native andselenomethionyl ODCase diffract to less than 2.2 AÊ and belong to theorthorhombic space group P212121, with unit-cell parameters a = 90.1,b = 116.2, c = 117.0 AÊ . Crystals of ODCase grown in the presence ofthe postulated transition-state analog inhibitor 6-hydroxyuridine50-phosphate (BMP) diffract to less than 2.5 AÊ and belong to spacegroup P21, with unit-cell parameters a = 79.9, b = 80.0, c = 98.2 AÊ , = 108.6.Received 5 October 1999Accepted 18 January 20001. IntroductionOrotidine 50-phosphate decarboxylase(ODCase; E.C. 4.1.1.23) catalyzes the ®nal stepin de novo pyrimidine biosynthesis yieldinguridine 50-phosphate (UMP), an essentialprecursor for synthesis of nucleic acids. Ineukaryotes, orotate phosphoribosyltransferase(OPRTase; E.C. 2.4.2.10) and ODCasecomprise separate catalytic domains of thebifunctional enzyme UMP synthase. However,in fungi and bacteria these enzymes exist asindependent monofunctional dimeric proteins(Yablonski et al., 1996). Compounds that areODCase inhibitors impair biosynthesis ofnucleic acids in all cell types and several havebeen tested as potential anticancer agentswithout success (Handschumacher et al., 1962;Sweeney et al., 1973). One of the most potentODCase inhibitors is 6-hydroxyuridine50-phosphate (BMP; Ki = 9  10ÿ12 M), acompound which resembles the postulatedintermediate generated in the transition stateduring OMP decarboxylation (Levine et al.,1980).The spontaneous loss of CO2 from OMP isan extremely slow reaction that proceeds witha half-time of nearly 7.8  107 y in aqueoussolutions at 298 K (Radzicka & Wolfenden,1995). Despite numerous studies involvingmodel decarboxylation reactions (Beak &Siegel, 1976; Silverman & Groziak, 1982; Lee& Houk, 1997), the mechanism employed byODCase remains a mystery. In contrast toother enzymatic decarboxylations, ODCase isunique in that it appears to function indepen-dently of known cofactors and metals (Shostak& Jones, 1992; Miller et al., 1999) whileenhancing the spontaneous rate of OMPdecarboxylation by nearly 17 orders ofmagnitude. Thus, structural studies of ODCaseprovide a source of new information thatshould enhance understanding of this enzyme'sreaction mechanism. Here, we describe crys-tallization conditions for recombinant yeastODCase, free and in complex with6-hydroxyuridine 50-phosphate, and presentinitial X-ray diffraction results for both thenative and selenomethionine-substitutedenzymes.2. Materials and methodsNative recombinant yeast ODCase wasexpressed in Escherichia coli strain SS6130 andFigure 1Photograph of native ODCase crystals grown at 277 K.Acta Cryst. (2000). D56, 472±474 Miller et al.  Orotidine 50-phosphate decarboxylase 473crystallization paperspuri®ed as described previously (Miller etal., 1999). Prior to its expression in E. coli,the AGA and AGG arginine codons of theyeast ura3 gene were changed to CGC orCGU. Stock solutions of puri®ed ODCase(18±25 mg mlÿ1) were stored at 253 K in50 mM Tris±HCl (pH 7.0) containing 5 mM-mercaptoethanol and 5% glycerol. Proteinconcentration was estimated from theabsorbance at 280 nm using a calculatedmolar extinction coef®cient of 28 830 cmÿ1(Edelhoch, 1967).Selenomethionyl ODCase was preparedby growing SS6130 (pBGM88) in a minimalmedia containing Vogel and Bonner salts(Vogel & Bonner, 1956), 0.002% vitamin B1,0.6% glycerol and 150 mg mlÿ1 ampicillin. Tothis solution, sterile amino acids were addedto give the following ®nal concentrations:dl-selenomethionine, 120 mg mlÿ1; alanine,glycine, serine and threonine, 0.5 mg mlÿ1;arginine, 0.6 mg mlÿ1; asparagine, aspartate,glutamine and lysine, 0.4 mg mlÿ1; cysteine,0.03 mg mlÿ1; glutamate, 0.7 mg mlÿ1; histi-dine and proline, 0.1 mg mlÿ1; isoleucine,leucine and valine, 0.25 mg mlÿ1; phenyl-alanine, 0.15 mg mlÿ1; tryptophan and tyro-sine, 0.2 mg mlÿ1. The expression cultureswere inoculated to an initial OD600 of 0.005and incubated at 300 K for 19±24 h, afterwhich the OD600 was 4.5. ODCase frombacteria grown in selenomethionine mediumwas puri®ed to greater than 95% homo-geneity using the same conditions employedfor puri®cation of the native protein.Analysis of the selenomethionyl enzyme byelectrospray mass spectrometry showed thatall six methionine residues per subunit werereplaced by selenomethionine.Crystals of native ODCasewere grown at 277 K by thehanging-drop vapor-diffusionmethod using 0.1 M Na HEPESpH 7.0, 10%(v/v) 2-propanol and20%(w/v) polyethylene glycol4000 as the precipitant. Theprotein drops were prepared byadding 2 ml of ODCase solutionto 3 ml of the reservoir solution.Crystals of native protein typi-cally appeared overnight. Prior todata collection, the crystals wereequilibrated in a cryoprotectantcomposed of 0.085 M Na HEPESpH 7.0, 8.5%(v/v) 2-propanol,17%(w/v) polyethylene glycol4000, 15% glycerol (HamptonCryo #41). Crystals of seleno-methionine-substituted ODCasewere grown as described for thenative enzyme except that thesecrystals required incubation for6±10 d at 277 K. Diffraction data for nativecrystals were collected at 93 K on a RigakuRU-200 rotating-anode generator equippedwith an MSC R-AXIS 4 area detector.Crystals of ODCase grown in thepresence of BMP were obtained at 295 K in0.18 M NaH2PO4, 18%(w/v) PEG 3350. Thehanging drops contained 8 mg mlÿ1 enzymeand BMP added in a 2.7:1 molar ratio.Crystals generally appeared after 7±10 d andwere equilibrated in cryoprotectantcontaining 0.18 M NaH2PO4, 18%(w/v)PEG 3350, 25%(v/v) glycerol prior to datacollection.3. Results and discussionScreening for optimal crystallization condi-tions was performed using sparse-matrixsampling (Jancarik & Kim, 1991; CrystalScreens I and II, Hampton Research) attemperatures in the range 277±295 K. Singlecrystals of native recombinant yeastODCase (0.74  0.66  0.37 mm) wereobtained from hanging drops incubatedovernight at 277 K (Fig. 1). The diffractionpattern shown in Fig. 2 represents a typical10 min, 1.5 oscillation image obtained withcrystals of native enzyme. These crystalsdiffracted to less than 2.0 AÊ resolution, wereorthorhombic and belonged to the spacegroup P212121, with unit-cell parametersa = 90.1, b = 116.2, c = 117.0 AÊ . Assuming aprotein density of 2.3 AÊ Daÿ1 and a solventcontent of 40%, there are four molecules perasymmetric unit (Matthews, 1968). Ingeneral, ODCase crystals remained stableon storage at 277 K for six months withoutloss in resolution.Replacement of all six ODCase methio-nine residues with selenomethionine did notappear to change the physical properties ofthis protein. The selenomethionyl enzymehad a speci®c activity that did not differnoticeably from the activity of the nativeenzyme. Crystals of the selenomethionylprotein were grown under the same crys-tallization conditions as de®ned for thenative enzyme. However, these crystals weremuch slower to develop and were markedlysmaller than those of the native enzyme.Attempts to increase crystal size by micro-seeding or to obtain crystals under differentconditions were not successful.The mechanism of enzymatic OMPdecarboxylation has to date eluded de®ni-tion. Our initial approach to the study of thisreaction involves de®ning those enzymeresidues that might participate in catalysisowing to their juxtaposition to the proposedtransition-state analog inhibitor BMP(Ki = 9  10ÿ12 M; Brody & Westheimer,1979). To this end, structural studies wereinitiated on ODCase complexed with BMP.Crystals of ODCase (270 mM in subunits)grown in the presence of excess BMP(740 mM) belonged to the space group P21,with unit-cell parameters a = 79.9, b = 78.0, c= 98.2 AÊ ,  = 108.6, suggesting that thesecrystals represent the enzyme±ligandcomplex. These crystals diffracted to lessthan 2.5 AÊ and contained four ODCasemonomers per asymmetric unit.Our ability to grow ODCase crystals wasaided by the original crystallization studiesof yeast ODCase complexed with BMP (Bellet al., 1991); however, the crystallizationconditions and crystal space groups differeddramatically between the two studies. Thislack of similarity may be because of physicaldifferences between ODCase puri®ed fromyeast and the recombinant yeast enzymeexpressed in and puri®ed from bacteria(Miller et al., 1999). Diffraction datacollected for selenomethionyl ODCase andthe native enzyme complexed with BMP arecurrently being analyzed. The ability togenerate diffraction-quality crystals offershope that the structures of free and ligandedODCase will be determined, therebyproviding an understanding of the forcesresponsible for this enzyme's remarkablecatalytic capabilities.We thank Jennifer Neugebauer forconducting mass-spectrometric analysis ofnative and selenomethionine-substitutedODCase. This work was supported in part byNIH Training Grant GM-08570.Figure 2A 1.5 oscillation image of a crystal of native ODCase. The crystalsdiffract nominally to 2.0 AÊ .474 Miller et al.  Orotidine 50-phosphate decarboxylase Acta Cryst. (2000). D56, 472±474crystallization papersReferencesBeak, P. & Siegel, B. M. (1976). J. Am. Chem. Soc.98, 3601±3606.Bell, J. B., Jones, M. E. & Carter, C. W. Jr (1991).Proteins, 9, 143±151.Brody, R. S. & Westheimer, F. H. (1979). J. Biol.Chem. 254, 4238±4244.Edelhoch, H. (1967). Biochemistry, 6, 1948±1954.Handschumacher, R. E., Calabresi, P., Welch,A. D., Bono, V., Fallon, H. & Frei, E. (1962).Cancer Chemother. Rep. 21, 1±18.Jancarik, J. & Kim, S.-H. (1991). J. Appl. Cryst. 24,409±411.Lee, J. K. & Houk, K. N. (1997). Science, 276,942±945.Levine, H. L., Brody, R. S. & Westheimer, F. H.(1980). Biochemistry, 19, 4993±4999.Matthews, B. W. (1968) J. Mol. Biol. 33, 491±497.Miller, B. G., Smiley, J. A., Short, S. A. &Wolfenden, R. (1999). J. Biol. Chem. 274,23841±23843.Radzicka, A. & Wolfenden, R. (1995). Science,267, 90±93.Shostak, K. & Jones, M. E. (1992). Biochemistry,31, 12155±12161.Silverman, R. B. & Groziak, M. P. (1982). J. Am.Chem. Soc. 104, 6434±6439.Sweeney, M. J., Davis, F. A., Gutowsk, G. E.,Hamill, R. L., Hoffman, D. H. & Poore, G. A.(1973). Cancer Res. 33, 2619±2623.Vogel, H. J. & Bonner, D. M. (1956). J. Biol. Chem.218, 97±103.Yablonski, M. J., Pasek, D. A., Han, B.-D., Jones,M. E. & Traut, T. W. (1996). J. Biol. Chem. 271,10704±10708.

Crystallization of native and selenomethionyl yeast orotidine 5′-phosphate decarboxylase

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Crystallization of native and selenomethionyl yeast orotidine 5′-phosphate decarboxylase

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