Acta Cryst. (2003). D59, 943±945 Huang et al. Imidase
943
crystallization papers
Acta Crystallographica Section D
Biological Crystallography
ISSN 0907-4449
Crystallization and preliminary X-ray diffraction
analysis of thermophilic imidase from pig liver
Cheng-Yang Huang,aSheng-Kuo Chiang,bYuh-Shyong Yanga* and Yuh-Ju Sunb*
aDepartment of Biological Science and
Technology, College of Science, National Chiao Tung University, Hsinchu, Taiwan, and
bInstitute of Bioinformatics and Structural
Biology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan
Correspondence e-mail: ysyang@cc.nctu.edu.tw, yjsun@life.nthu.edu.tw
# 2003 International Union of Crystallography Printed in Denmark ± all rights reserved
Imidase is an enzyme, also known as dihydropyrimidinase (EC 3.5.2.2), hydantoinase, dihydropyrimidine hydrase or dihydropyrimi-dine amidohydrolase, that catalyzes the reversible hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate and many other imides. Substrate speci®city, metal content and amino-acid sequence all differ signi®cantly between bacterial and mammalian imide-hydrolyzing enzymes. In this study, a thermophilic imidase was isolated from pig liver and crystallized. Two kinds of imidase crystals were grown by the hanging-drop vapour-diffusion method using polyethylene glycol MME 5000 and 2-propanol as precipitants. One belongs to the triclinic P1space group, with unit-cell parameters a = 96.35, b = 96.87, c = 154.87 AÊ, = 82.10, = 72.54, = 77.19, and the other belongs to the orthorhombic C2221 space group, with unit-cell parameters a = 113.92, b = 157.22, c = 156.21 AÊ.
Received 26 November 2002 Accepted 12 March 2003
1. Introduction
Imide-hydrolyzing enzymes (EC 3.5.2.2) were ®rst described in the 1940s as hydrolyzing hydantoin derivatives in plants and animals (Bernheim & Bernheim, 1946; Eadie et al., 1949). The natural substrate of an imide-hydrolyzing enzyme isolated from calf liver was shown to be dihydropyrimidine and it was therefore named dihydropyrimidinase (Wallach & Grisolia, 1957). A detailed inves-tigation into enzyme substrates showed that the hydrolytic cleavage of imides catalyzed by the rat liver enzyme ranges from linear imides to heterocyclic imides and includes hydantoins, dihydropyrimidines and phthalimide. There-fore, the imide-hydrolyzing enzyme was classi®ed as an imidase (Yang et al., 1993). In vitro data indicate that imidase prefers xeno-biotics to physiological substrates (Yang et al., 1993). The substrate spectrum of imidase was further shown to include the cyclic carbonates (Yang et al., 1998). A recent ®nding shows that the imidase from pig liver is thermophilic. The optimum temperature for catalysis of this mammalian enzyme is more than 20 K higher than that of its native source and its substrate speci®city is temperature dependent (Su & Yang, 2000).
All known mammalian imidases are tetra-mers that contain four tightly bound Zn atoms, with one Zn atom per subunit (Brooks et al., 1983; Kautz & Schnackerz, 1989; Kikugawa et al., 1994; Huang & Yang, 2002). However, variations are found in the structure, metal content, substrate speci®city and other requirements of similar enzymes from different organisms. Imidase from Bacillus stearo-thermophilus SD-1 is a dimer and contains
manganese, with one Mn atom per dimer (Lee et al., 1995). Imidase from Blastobacter sp. A17p-4 is a trimer (Ogawa et al., 1997). Imidase from Pseudomonas putida 77 requires ATP for its amidohydrolysis (Ogawa et al., 1995). Imidase from Arthrobacter aurescens DSM 3745 is a zinc enzyme (May et al., 1998a) and contains 10 moles of zinc per mole of enzyme (May et al., 1998b).
Recently, the crystal structures of bacterial enzymes from Thermus sp. (Abendroth, Nie®nd & Schomburg, 2002; Abendroth, Nie®nd, May et al., 2002) and B. stearo-thermophilus (Cheon et al., 2002) were resolved. They were all shown to contain bridged dimetal ions in the enzyme subunit. It has also been reported that some bacterial imide-hydrolyzing enzymes are able to hydro-lyze sulfur-containing cyclic imides, simple cyclic imides and simple cyclic ureides, but that substrates do not include substituted cyclic ureides and bulky substituted cyclic imides (Ogawa et al., 1997). It would be interesting to discover how nature has evolved such varied enzyme systems for imide hydrolysis in different organisms. Since the crystal structure of the bacterial enzyme is available, further structural analysis of mammalian imidase will allow the difference between these imidases to be better understood. Here, we report the ®rst preliminary analysis of a mammalian imidase crystal.
2. Materials and methods
Pig liver imidase was puri®ed as described previously (Su & Yang, 2000) with some modi®cations to obtain extra high purity for
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Huang et al. Imidase Acta Cryst. (2003). D59, 943±945crystallization papers
crystallization. The thermophilic pig liver imidase was concentrated to 30 mg mlÿ1in 20 mM Tris±HCl. Crystallization trials were set up by the hanging-drop vapour-diffusion method (McPherson, 1982). 2 ml protein samples were equilibrated against a 500 ml reservoir solution in Linbro plates. Initial crystallization conditions were obtained using Hampton Research Crystal Screen kits (Hampton Research, California, USA) and then further optimized to obtain diffraction-quality crystals.
X-ray diffraction data from form A crys-tals (Fig. 1a) were measured at 0.9537 AÊ using a Quantum-4R CCD detector on the BL12B2 Taiwan beamline at SPring-8, Japan. The X-ray diffraction data from form B crystals (Fig. 1b) were collected on a Rigaku R-AXIS IV++ image-plate detector using Cu K radiation from a Rigaku RU-300 rotating-anode X-ray generator operated at 50 kV and 100 mA. Thermo-philic pig liver imidase form A crystals were grown from 10%(w/v) polyethylene glycol MME 5000; extra cryoprotectant was needed for data collection at low tempera-tures (100 K). The optimized cryoprotectant buffer prevented ice-ring formation in the ¯ash-frozen crystal. Firstly, the crystals were transferred directly into a cryoprotectant buffer containing 15%(v/v) glycerol and incubated for 30 min before data collection. Higher concentrations of glycerol (>15%) melted the crystal. The form B crystal was
directly frozen in liquid nitrogen immedi-ately before data collection without using any extra cryoprotectant reagent. Crystals were mounted in a nylon loop and ¯ash-frozen in a nitrogen stream (Oxford Cryo-systems). The programs DENZO and SCALEPACK (Otwinowski & Minor, 1997) were used for the X-ray diffraction data processing and analysis.
3. Results
Two forms of crystal were grown under quite different crystallization conditions using
polyethylene glycol MME 5000 and 2-propanol as precipitants. Needle-shaped form A crystals (Fig. 1a) grew in 3±5 d to maximum dimensions of 0.05 0.05 0.8 mm using 10%(w/v) polyethylene glycol MME 5000, 100 mM sodium chloride, 100 mM bicine buffer pH 9.0 and 2%(w/v) 1,6-hexanediol. Form B crystals (Fig. 1b) grew over a longer period of time, 3±4 weeks, to maximum dimensions of 0.05 0.08 0.1 mm using 30%(v/v) 2-propanol, 0.2 M sodium citrate and 100 mM sodium cacodylate pH 6.5. Form A crystals diffracted to 2.2 AÊ and belonged to the
Figure 1
Photographs showing crystals of thermophilic pig liver imidase. (a) Form A crystals, triclinic P1 space group. (b) Form B crystals, orthorhombic C2221space
group.
Table 1
Summary of X-ray diffraction data statistics.
Values in parentheses are for the outermost resolution shell.
Form A Form B
Wavelength (AÊ) 0.9537 1.54
Crystal system Triclinic Orthorhombic
Space group P1 C2221
Unit-cell parameters
a, b, c (AÊ) 96.35, 96.87, 154.87 113.91, 157.30, 156.26 , , () 82.10, 72.54, 77.19 90, 90, 90
Resolution (AÊ) 2.2 3.0
No. of re¯ections collected 555227 92506
No. of unique re¯ections 253875 53857
Redundancy of re¯ections 1.8 1.72
Data completeness (%) 96.2 (92.7) 99.2 (99.9)
I/(I) 8.04 (1.84) 6.3 (3.5)
Rmerge(%) 9.3 (46) 11.9 (20.9)
Figure 2
Acta Cryst. (2003). D59, 943±945 Huang et al. Imidase
945
crystallization papers
triclinic space group, with unit-cell para-meters a = 96.35, b = 96.87, c = 154.87 AÊ, = 82.10, = 72.54, = 77.19(Table 1). Form B crystals diffracted to 3.0 AÊ and belonged to the orthorhombic space group C2221, with unit-cell parameters a = 113.92, b = 157.22, c = 156.21 AÊ (Table 1).
Most of the imidases are described in the literature as forming tetramers and a few as forming dimmers or trimers, based on gel chromatography and dynamic light-scattering studies. The molecular weight for a single polypeptide chain of pig liver imidase was estimated to be around 51 kDa. However, the molecular weight of the quaternary structures of pig liver imidase was about 300 kDa as determined by gel ®ltration. Thus, pig liver imidase may indeed function as a tetramer (Su & Yang, 2000). In the P1 triclinic crystal, we predicted there to be 12 molecules (three tetramers) per asymmetric unit in the crystal packing from the VM (Matthews, 1968) constant
calcula-tion. The VM (Matthews, 1968) was
calculated to be 2.26 AÊ3Daÿ1 and the solvent content was estimated to be 46%. The self-rotation function was calculated using the GLRF program (Tong & Rossmann, 1990). The non-crystallographic symmetric relationships of molecules are shown at angles of 180, 120, 90 and 60in Fig. 2. Pseudo twofold, threefold, fourfold and sixfold axes were found. For the C2221 orthorhombic crystal, the VM (Matthews, 1968) was calculated to be 2.29 AÊ3Daÿ1, corresponding to a solvent content of 47%, assuming three molecules per asymmetric unit in the crystal. Pseudo twofolds and threefold axes were found from rotation function calculations. The self-rotation function peaks are shown at angles of 180 and 120 in Fig. 3. Structure determination by the molecular-replace-ment method using the imidases of Thermus sp. (Abendroth, Nie®nd & Schomburg, 2002) and B. stearothermophilus (Cheon et al., 2002) as search models is also in progress.
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