PROTEINS: Structure, Function, and Genetics 26:118-120 (1996)

Crystallization and Preliminary X-Ray Diffraction Studies of Formylmethanofuran: Tetrahydromethanopterin Formyltransferase From Methampyrus kandleri Seigo Shima,' Rudolf K. Thauer,' Hartmut Michel? and Ulrich Ermlel.2 lMez-Planck-Institut fur terrestrische Mikrobiologie and Laboratorium f i r Mikrobiologie der Philipps-Universitat, 035043 Marburg, Germany, 2Ma.x-Planck-Znstitut fur Biophysik, 0-60528 Frankfurt, Germany

ABSTRACT Formy1methanofuran:tetra- hydromethanopterin formyltransferase from the hyperthermophilic methanogenic Archaeon Methampyrus kandleri (growth temperature optimum 98°C) was crystallized by vapor diffu- sion methods. Crystal form M obtained with 2-methyl-2,4-pentanediol as precipitant dis- played the space group P2, with unit cell pa- rameters o f a = 87.0A, b = 75.4hi,c = 104.7A, and f3 = 113.9" and diffracted better than 2 hi resolution. Crystal form P grown from polyeth- ylene glycol 8000 belonged to the space group 14'22 and had unit cell parameters of 157.5 A and 242.1 hi. Diffraction data to 1.73 A were re- corded. Crystal form S which was crystallized from (NH,),SO, in the space group 14'22 with unit cell parameters of 151.3 A and 249.5 A dif- fracted at least to 2.2 hi resolution. All crystal forms probably have four molecules per asym- metric unit and are suitable for X-ray structure analysis. o 1996 Wiley-Liss, hc.

Key words: Protein crystallization, X-ray crys- tallography, methanogenic Ar- chaea, hyperthermophilic en- zymes, halophilic enzymes

Formy1methanofuran:tetrahydromethanopterin formyltransferase (Ftr) catalyzes the reversible conversion of formylmethanofuran and tetra- hydromethanopterin to methanofuran and N5- formyltetrahydromethanopterin.' Ftr is found in methanogenic and sulfate-reducing Archaea. In these strictly anaerobic microorganisms it is in- volved in CO, reduction to CH,, in autotrophic CO, fixation, in C,-unit formation from C02 and/or in C,-unit oxidation to C0,.2-4

Ftr is also present in Methanopyrus kandleri, which is an abyssal hyperthermophilic Archaeon growing optimally a t 98°C on H, and CO, with the formation of methane.5,6 The organism, which has a G + C content of 60 mol%, is phylogenetically only distantly related to all other known methanogen~.~

0 1996 WILEY-LISS, INC.

The Methanopyrales represent as yet the deepest branch in the kingdom of Euryarchaeota.'

Ftr from M. kandleri is a soluble monomeric en- zyme of molecular mass 32 kDa, which is stable un- der oxic conditions. It is devoid of a chromophoric prosthetic group and exhibits a ternary-complex type catalytic mechani~m.~ The enzyme is absolutely de- pendent on the presence of lyotropic salts for activity, the concentration dependence being sigmoidal. For maximal activity 1.5-2 M of K,HPO, or (NH,),SO, are required. The presence of lyotropic salts is also required for thermostability of the enzyme, maximal thermostability being observed at 1.5 M concentra- tion of K,HPO, at pH 8.0. The salt requirement for catalytic activity and thermostability probably re- flects that the intracellular concentration of salts in M. kandleri is very high, the predominant solutes being cyclic 2,3-diphosphoglycerate and potassium ions, both present a t concentrations above 1 M.5,6

The primary structure of Ftr from M. kandleri has been determined and compared with those of Ftr from thermophilic and mesophilic methanogens." The enzyme from M. kandleri was found to have the lowest isoelectric point and the lowest hydrophobic- ity of the amino acid composition. The relatively low hydrophobicity may reflect that Ftr from M. kandleri is adapted not only to hyperthermophilic but also to the halophilic conditions prevailing within the

Crystal structures of only four enzymes from hy- perthermophilic organisms have, until now, been de- termined: a1dehyde:ferredoxin oxidoreductase from Pyrococcus furiosus (growth temperature optimum 100°C),16 glutamate dehydrogenase from P. furio- SUS,'~ glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima (80°C),18 and indol-3- glycerol phosphate synthase from Sulfolobus solfa- taricus (87"C)." Structure comparisons revealed

Received February 23, 1996; revision accepted March 12, 1996.

Address reprint requests to Dr. Ulrich Ermler, Max-Planck- Institut fur Biophysik, Heinrich-Hoffmann-Strde 7, D-60528 Frankfurt, Germany.

FORMYLTRANSFERASE FROM METHANOPYRUS KANDLERI 119

that clusters of salt bridges may be an important factor contributing to the high thermostability of the proteins. The crystal structure of only one halophilic enzyme has been determined, namely, that of malate dehydrogenase from the halophilic Archaeon Ha- loarcula marismortui. Excess of acidic over basic res- idues distributed on the enzyme surface and salt bridges appear to be important for the stability of the enzyme."

Methanogenesis is a unique biological pathway involving novel coenzymes and enzymes.'l The structure of all the coenzymes have, in the mean- time, been elucidated. Structural analysis of meth- anogenic enzymes has only recently been initiated. In this communication we report for the first time the crystallization of a methanogenic enzyme.

Ftr from M. kandleri was produced by the expres- sion of the fir gene in Escherichia coli BL21 (DE3) under the control of T7 promoter with subsequent purification, as described previously, with some modifications." Column chromatography and con- centration of the enzyme were performed at 4°C un- der oxic conditions. Protein was determined by the method of Bradford" using the reagents from Bio- Rad Laboratories and with ovalbumin as standard. The purified enzyme solution was diluted by 50 mM Mops pH 7 containing 2 mM dithiothreitol and con- centrated to 12-16 mg/ml by ultrafiltration, using 10 ml Omegacell stirred-cell devices (Filtron GmbH, Karistein, Germany).

Crystallization experiments were performed with the hanging drop vapor diffusion method using a sparse matrix crystallization screening kit (Hamp- ton Research, Laguna Hills, CA) described by Jan- carik and Kimz3 for initial screening. Three success- ful crystallization conditions were found and improved by systematic search. The obtained crys- tals, form M, P, and S, were analysed on a Rigaku RU-200 X-ray generator with CuK, radiation using a MAR-Research imaging plate as detector.

Best crystals of form M were grown by mixing 2 p1 enzyme solution and 2 pl reservoir solution consist- ing of 20% 2-methyl-2,4-pentanediol, 0.7 M NaC1, and 0.1 M sodium acetate, pH 4.6, on the drop. The drop was equilibrated against 1 ml of reservoir so- lution at a temperature of 4°C. Plate-shaped crys- tals, which were observed within 2 weeks, reached a maximum size of 0.1 x 0.3 x 0.6 mm3 and dif- fracted beyond a resolution of 2 A. They grew in the space group P2, and their unit cell dimensions were a = 87.0 A, b = 75.4 A, c = 104.7 A, and p = 113.9' determined by autoindexing oscillation photographs with the program REFIX.', The presence of four molecules per asymmetric unit gives a crystal vol- ume per protein mass (vM) of 2.5 ii3/Da,25 implying a solvent content of 50%. Three or five molecules per asymmetric unit are less probable, but the resulting V, value would fall also in the range observed for water-soluble proteins. The crystal form M exhib-

ited a relatively high mosaicity and a limited life- time under the X-ray beam.

Crystal form P was obtained at 4"C, using 4 pl enzyme solution and 4 p1 reservoir solution, which contains 22% polyethylene glycol (PEG) 8000,0.3 M (NH,),SO,, and 0.1 M Mops, pH 7.0. Bipyramidal crystals appeared reproducibly within 2 weeks and grew to a size of 0.6 x 0.6 x 0.8 mm3. They dif- fracted to 1.7 A resolution and belonged to the space groupZ4,22 with unit cell parameters of a = 157.5 A and c = 242.1 A. Assuming four molecules per asymmetric unit the V, value was 3.0 A3/Da, and the solvent content was 58%. The VM value calcu- lated with five or six molecules per asymmetric unit would also be compatible with normally found crys- tal packing densities.

Best crystals of crystal form S were grown to a size of 0.4 x 0.5 x 0.5 mm3 when the reservoir solution was composed of 1.2 M (NH,),SO,, 1% PEG 8000, 0.1 M sodium citrate, pH 5.6. The obtained rhombo- hedral crystals diffracted at least to a resolution of 2.2 A. The space group was determined to be I4,22 and the unit cell parameters to be a = 151.3 A and c = 249.5 consistent with four, five, or six mole- cules in the asymmetric unit. The identical space group and the similar unit cell dimensions of crystal forms P and S suggest a related crystal packing.

Native data were collected from three crystals of crystal form P using synchrotron radiation of the Max-Planck beamline at DESY (Deutsches Elek- tronensynchrotron, Hamburg). The intensities were evaluated with the program MOSFLMZ6 and inter- nally scaled with programs of the CCP4 pa~kage.'~ 408,443 reflections were measured and reduced to a final data set of 137,108 unique reflections with an Rsym value of 5.4%. This represents a completeness of 87.7% in the resolution range from 1.73 to 30.0 A. Structure determination by multiple isomorphous replacement techniques is in progress.

ACKNOWLEDGMENTS We thank Heidi Muller for technical assistance

and the staff of the Max-Planck beamline at the Deutsches Elektronensynchrotron in Hamburg for help in data collection.

REFERENCES 1. Donnelly, M.I., Wolfe, R.S. The role of formylmetha-

n0furan:tetrahydromethanopterin formyltransferase in methanogenesis from carbon dioxide. J. Biol. Chem. 261: 16653-16659, 1986.

2. Wolfe, R.S. My kind of biology. Annu. Rev. Microbiol. 45: 1-35,1991.

3. Thauer, R.K., Hedderich, R., Fischer, R. Biochemistry. Re- actions and enzymes involved in methanogenesis from CO, and H,. In: "Methanogenesis." Ferry, J.G. (eds.). New York: Chapman & Hall, 1993:209-252.

4. Vorholt, J., Kunow, J., Stetter, K.O., Thauer, R.K. En- zymes and coenzymes of the carbon monoxide dehydroge- nase pathway for autotrophic CO, fixation in Archaeoglo- bus lithotrophicus and the lack of carbon monoxide

120 S. SHIMA ET AL.

dehydrogenase in the heterotrophic A. profundus. Arch. Microbiol. 163:112-118, 1995.

5. Huber, R., Kurr, M., Jannasch, H.W., Stetter, K.O. A novel group of abyssal methanogenic archaebacteria (Methano- pyrus) growing at 110°C. Nature 342:833-834, 1989.

6. Kurr, M., Huber R., Konig, H., Jannasch, H.W., Fricke, H., Trincone, A., Kristjansson, J.K., Stetter, K.O. Methanopy- rus kandleri, gen. and sp. nov. represents a novel group of hyperthermophilic methanogens, growing at 110°C. Arch. Microbiol. 156239-247, 1991.

7. Burggraf, S., Stetter, K.O., Rouvihre, P., Woese, C.R. Methanowrus kandleri: An archaeal methanogen unre- lated to Ail other known methanogens. System.ipp1. Mi- crobiol. 14:346-351, 1991.

8. Woese, C.R., Kandler, O., Wheelis, M.L. Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. U.S.A. 87: 4576-4579, 1990.

9. Breitung, J., Borner, G., Scholz, S., Linder, D., Stetter, K.O., Thauer, R.K. Salt dependence, kinetic properties and catalytic mechanism of N-formy1methanofuran:tetrahy- dromethanopterin formyltransferase from the extreme thermophile Methunopyrus kandleri. Eur. J. Biochem. 210: 971-981,1992,

10. Shima, S., Weiss, D.S., Thauer R.K. Formylmethanofuran: tetrahydromethanopterin formyltransferase (Ftr) from the hyperthermophilic Methanopyrus kandleri. Cloning, se- quencing and functional expression of the ftr gene and one-step purification of the enzyme overproduced in Es- cherichiu coli. Eur. J. Biochem. 230:906-913, 1995.

11. Menendez-Arias, L., Argos P. Engineering protein thermal stability. Sequence statistics point to residue substitutions in a-helices. J . Mol. Biol. 206:397-406, 1989.

12. Zwickl, P., Fabry, S., Bogedain, C., Haas, A., Hensel, R. Glyceraldehyde-3-phospate dehydrogenase from the hy- perthermophilic archaebacterium Pyrococcus woesez: Characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichiu coli. J . Bacteriol. 172:4329-4338,1990.

13. Jaenicke, R. Protein stability and molecular adaptation to extreme conditions. Eur. J. Biochem. 202:715-728, 1991.

14. Jaenicke, R. ZBvodszky, P. Proteins under extreme physi- cal conditions. FEBS Lett. 268:344-349, 1990.

15. Zaccai, G., Eisenberg, H. Halophilic proteins and the in- fluence of solvent on protein stabilization. Trends Bio- chem. Sci. 15333-337.1990.

16. Chan, M.K., Mukund, S., Kletzin, A., Adams, M.W.W., Rees, D.C. Structure of a hyperthermophilic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase. Science 267:

17. Yip, K.S.P., Stillman, T.J., Britton, K.L., Artymiuk, P.J., Baker, P.J., Sedelnikova, S.E., Eagel, P.C., Pasquo, A., Chiaraluce, R., Consalvi, V., Scandurra, R., Rice, D.W. The structure of Pyrococcus furiosus glutamate dehydroge- nase reveals a key role for ion-pair networks in maintain- ing enzyme stability at extreme temperatures. Structure 3:1147-1158, 1995.

18. Korndorfer, I., Steipe, B., Huber, R., Tomschy, A., Jaen- icke, R. The crystal structure of holo-glyceraldehyde-3- phosphate dehydrogenase from the hy erthermophilic bacterium Thermotoga maritima at 2.5 H resolution. J. Mol. Biol. 246:511-521, 1995.

19. Hennig, M., Darimont, B., Sterner, R., Kirschner, K., Jan- sonius J.N. 2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfatari- cus: Possible determinants of protein stability. Structure 3:1295-1306,1995,

20. Dym, O., Mevarech, M., Sussman, J.L. Structural features that stabilize halophilic malate dehydrogenase from an archaebacterium. Science 267:1344-1346, 1995.

21. Weiss, D.S., Thauer, R.K. Methanogenesis and the unity of biochemistry. Cell 72:819-822, 1993.

22. Bradford, M.M. A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248- 254,1976.

23. Jancarik, J., Kim, S.-H. Sparse matrix sampling: a screen- ing method for crystallization of proteins. J. Appl. Crys- tallogr. 24:409-411, 1991.

24. Kabsch, W. Automatic indexing of rotation diffraction pat- terns. J . Appl. Crystallogr. 2157-71, 1988.

25. Matthews, B.W. Solvent content of protein crystals. J . Mol. Biol. 33:491-497, 1968.

26. Leslie, A.G.W. Recent changes to the MOSFLM package for processing film and image plate data. In: “Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography, No. 26.” Warrington, UK: Daresbury Laboratory, 1992.

27. Collaborative Computational Project, Number 4. The CCP4 Suite: Programs for Protein Crystallography. Acta Crystallogr. D50760-763, 1994.

1463-1469,1995.