J. Mol. Biol. (1992) 226, 1279-1281


Crystallization and Preliminary Crystallographic Characterization of GTP Cyclohydrolase I from Escherichia colit Cornelia Schmid’, Rudolf Ladenstein**, Hartmut Luecke*, Robert Hubeti and Adelbert Bather’ ‘Lehrstuhl

fiir Organische Chemie und Biochemie, Technische Universitdt Miinchen Lichtenbergstr. 4, D-8046 Garching, Federal Republic of Germany 2Max-Planck-Institut D-8033 Martinsried, (Received 14 April

fiir Biochemie, Am Klopferapitz Federal Republic of Germany 1992; accepted 14 April


GTP cyclohydrolase I of Escherichia coli has been purified from a recombinant bacterial strain. The enzyme was crystallized from 0.6 M-sodium citrate and from 98 M-sodium/ potassium phosphate, respectively. Crystals grown in citrate showed X-ray diffraction extending to a resolution better than 3 A. The space group was P2, with cell dimensions a = 204.8 A, 6 = 210-l A, c = 72.2 A, a = y = 90” and /l = 95.8”. Keywords:


cyclohydrolase dihydrofolate;

I; crystallization; X-ray diffraction; biosynthesis of tetrahydrobiopterin

GTP cyclohydrolase (EC catalyzes a ring expansion of GTP (Fig. l(a)) conducive to the formation of dihydroneopterin triphosphate (Fig. l(b)) (Brown & Williamson, 1987; Yim $ Brown, 1976; Burg & Brown, 1968). The complex reaction starts with the release of formate from C-8 of the imidazole ring of the purine system by two consecutive hydrolytic steps. It is assumed that the ribose side-chain subsequently undergoes an Amadori reaction followed by ring closure involving the newly formed carbonyl group with the position 5 amino group of the pyrimidine ring. In microorganisms and plants, the product of GTP cyclohydrolase I serves as an intermediate in the biosynthesis of tetrahydrofolate (Fig. l(d)) (Brown & Williamson, 1987). In vertebrates, the enzyme product serves as an intermediate in the biosynthesis of tetrahydrobiopterin (Fig. l(c)) (Blau $ Niederwieser, 1986; Nichol et al., 1985). The gene for GTP cyclohydrolase I in Escherichia coli has been cloned (Katzenmeier et al., 1990) and has been shown to code for a polypeptide of 223 amino acids (Katzenmeier et al., 1991). The rat gene was cloned from a cDNA library and was shown to code for a 25 kDa protein consisting of 241 amino

7 This paper is dedicated

to Dr Irmgard

Ziegler on

the occasion of her birthday. $ Present address: Center for Structural Biochemistry, Karolinska Institute, S-14157 Huddinge, Sweden. 0022-2836/92/161279-03





acids (Hatakeyama et al., 1989, 1991). Despite their involvement in different biosynthetic pathways, these enzymes show about 50% sequence homology extending over the entire length of the proteins (Katzenmeier et al., 1991). A segment of the gene coding for human GTP cyclohydrolase I was cloned by a PCR (polymerase chain reaction) strategy based on the similarity between the genes from E. coli and rat (M. Giitlich, K. Schott, T. Werner, A. Bather & M. Ziegler, unpublished results). GTP cyclohydrolase I of E. coli has been purified to homogeneity by affinity chromatography (Yim & Brown, 1976). A molecular mass of 210 kDa has been proposed on the basis of gel filtration experiments, and an octamer structure has been proposed for the active enzyme (Yim & Brown, 1976). Enzymatically inactive dimers are observed in appropriate buffers. These findings suggest that the protein is a tetramer of dimers. The protein has a doughnut shape in electron micrographs of negative-stained specimens (Yim & Brown, 1976). GTP cyclohydrolase I was purified from the E. coli wild-type B DSM 613 (Deutsche Sammlung von Mikroorganismen, Braunschweig, Federal Republic of Germany) by the procedure of Yim & Brown (1976). A modified procedure was used for the purification of the enzyme from the recombinant strain E. co.&pCYH (Katzenmeier et al., 1990). Prior to crystallization experiments, the stability of the enzyme at room temperature was examined in the presence of various precipitating agents. The 0

1992 Academic




C. Schmid et al.

(01 0 -




Figure 1. Biosynthesis of pteridine derivatives. A, reaction catalyzed by GTP cyclohydrolase I.

enzyme showed excellent long-term stability in 1 M-ammonium sulfate containing 10 mM-phosphate and 25 mM-EDTA at pH 7.0. No loss of activity was observed during a period of ten months. The concentration of citrate required for protein precipitation depended strongly on pH (about 160 mM at pH 60 and 600 mM at pH 82). Optimum stability in citrate was found at around pH 7 with a half-life period of about six months. In 964 M-phosphate buffer at pH 7.5 to 80 the protein stability was limited, with a half-life period of two to three weeks. Crystallization experiments were subsequently initiated by the vapor diffusion method under experimental conditions affording appropriate protein stability. Crystals with the appearance of rectangular plates or prisms were obtained from 66 M-citrate in the range of pH 7.4 to 8.2. Frequently, crystals with an almost cubic appearance were observed (Fig. 2). The largest crystals observed were up to 94 mm across. Amorphous precipitates usually formed prior to the appearance of the crystals that grew subsequently from the turbid solution. Crystal growth was slow, and frequently crystals were only obtained after seeding with macroscopic crystal fragments. Crystals were also formed from 08 M-phosphate at pH 7.5 and 69 M-phosphate at pH 8.0. The crystallization tendency was higher than in citrate, and seeding was not required. The crystals appeared with a variety of polygonal shapes. Prisms with a pentagonal cross-section were observed frequently. Small wedge-shaped or polygonal crystals were obtained from 50 mM-potassium phosphate (pH 7-O), containing 6.7 y. polyethylene glycol 8000. Only very small, needle-shaped crystals were formed in ammonium sulfate. X-ray precession and rotation photographs of the crystal forms obtained from citrate buffer and phosphate buffer were recorded on Huber cameras (Huber, Rimsting, Federal Republic of Germany) in a thermostatically controlled room at 15°C. The X-ray source was a Rigaku RU 200 rotating-anode generator operating at 45 kW. The focal spot size was 63 mm x 30 mm. Graphite-monochromatized CuKu radiation was used. The crystal-to-focus/ crystal-to-film distances were 300 mm/100 mm. The

Figure 2. Crystals of GTP cyclohydrolase I from a recombinant strain of E. coli K12 grown at 20°C in 66 M-sodium citrate (pH 7.4), containing 20 mMphosphate, 5 mM-EDTA and 006% sodium azide.

exposure time for rotation photographs was about 20 hours/degree of rotation. X-ray diffraction studies were conducted with protein crystals grown in 660 M-citrate (pH 7.4). Crystals of the enzyme from E. coli B wild-type diffracted X-rays to about 3.4 A (1 A = 61 nm) resolution on still photographs. Crystals of the enzyme from the recombinant E. coli K12 strain diffracted to a resolution substantially better than 3.0 A. The examination of screenless precession and rotation photographs demonstrated 2/m Laue symmetry. The photographs showed systematic absences of hkl reflections when 1= 2n+ 1, those extinctions that characterize space group P2, (Fig. 3). The monoclinic unit cell has dimensions of a = 2048 A, b = 210.1 A, c = 72-2 8, u = y = 90” and /l = 958”. From the distribution of the reflection intensities and the unit cell constants, tetragonal pseudo-symmetry with the 4-fold axis approximately along c is obvious. On the basis of the unit cell volume of V = 3,090,752 A3, one can conclude that the asymmetric unit of the crystal cell might be occupied by two, three or four cyclohydrolase octamers (M, = 24,870), resulting in packing densities of 3.8 A3/Da, 2.6 A3/Da and 1.9 A3/Da, respectively, and indicating a rather large asymmetric unit in the citrate modification of cyclohydrolase I. With the analysis of the non-crystallographic symmetry, on the other hand, real space averaging of the electron density maps would be possible, providing valuable information for phase determination. In light of the sequence homology between the bacterial and the vertebrate enzymes, it appears likely that the bacterial enzyme could serve as a structural and mechanistic model for mammalian GTP cyclohydrolase I.


F Figure 3. Rotation photograph of GTP cyclohydrolase O= 0 to 3”; crystal-to-film distance, 100 mm; collimator,

I. The crystal was grown in citrate buffer (see Fig. 1). hk pla 64 mm; exposure time, 10 h/deg.

This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. We thank Angelika Kohnle for help with the preparation of the manuscript.

References Blau, N. & Niederwieser, A. (1985). GTP-cyclohydrolases: a review. J. Clin. Chem. Clin. Biochem. 23, 169-176. Brown, G. M. & Williamson, J. M. (1987). Biosynthesis of folic acid, riboflavin, thiamine, and pantothenic acid. In Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology (Neidhardt, F. C., ed.). vol. 1, pp. 521-538, American Society for Microbiology, Washington, DC. Burg, A. W. & Brown, G. M. (1968). The biosynthesis of folic acid. VIII. Purification and properties of the enzyme that catalyzes the production of formate from carbon atom 8 of guanosine triphosphate. J. Biol.





binding of GTP to the enzyme. J. Biol. 21660-21664. Hatakeyama, K., Inoue, Y., Harada, T. & Kagamiyama, H. (1991). Cloning and sequencing of cDNA encoding rat GTP cyclohydrolase I. The first enzyme of the tetrahydrobiopterin biosynthetic pathway. J. Biol. Chem.





Katzenmeier, G., Schmid, C. & Barher. A. (1990). Cloning and expression of the putative gene coding for GTP cyclohydrolase I from Escherichia coli. FEMS Microbial. Letters, 66, 231-234. Katzenmeier, G., Schmid, C., Kellermann, J., Lottspeich, F. & Bather, A. (1991). Biosynthesis of tetrahydrofolate. Sequence of GTP cyclohydrolase I from Escherichia






991-997. Nichol, C. A., Smith, G. K. & Duch, D. S. (1985). Biosynthesis and metabolism of tetrahydrobiopterin Annu. Rev. Biochem. 54. and molybdopterin.



Hatakeyama, K., Harada, T., Suzuki, S., Watanabe, Y. & Kagamiyama, H. (1989). Purification and characterization of rat liver GTP cyclohydrolase I. Edited


J. J. & Brown, G. M. (1976). Charact*eristics of guanosine triphosphate cyclohydrolase purified from Escherichia coli. J. Biol. Chem. 251. 5087-5094.

by A. Klug

Crystallization and preliminary crystallographic characterization of GTP cyclohydrolase I from Escherichia coli.

GTP cyclohydrolase I of Escherichia coli has been purified from a recombinant bacterial strain. The enzyme was crystallized from 0.6 M-sodium citrate ...
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