Protein Preparation, Crystallization and Preliminary X-Ray Analysis of Imidazolonepropionase from Bacillus Subtilis

http://www.paper.edu.cn 中国科技论文在线

Biochimica et Biophysica Acta 1764 (2006) 153 – 156

Short crystallization paper Protein preparation, crystallization and preliminary X-ray analysis of imidazolonepropionase from Bacillus subtilis

Yamei Yu, Lanfen Li, Xiaofeng Zheng, Yu-He Liang, Xiao-Dong Su *

The National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, P.R. China

Received 25 June 2005; received in revised form 2 August 2005; accepted 3 August 2005 Available online 12 September 2005

Abstract

Imidazolonepropionase (EC 3.5.2.7) is the third enzyme of the histidine degradation pathway that has been conserved from bacteria to eukaryotes. The enzyme is the only one with unknown three-dimensional structure in this pathway. In this work, Bacillus subtilis imidazolonepropionase (HutI) was expressed in E. coli and purified to homogeneity. After thrombin digestion, high quality crystals were obtained by hanging-drop vapor diffusion method. The best crystal diffracted to 2.0 A˚ and belonged to the space group P21 with unit-cell parameters a =57.73 A˚ , b =106.34 A˚ , c =66.47 A˚ , b =89.93-. D 2005 Elsevier B.V. All rights reserved.

Keywords: Imidazolonepropionase; Crystallization; Thrombin-digestion; X-ray diffraction

Although higher animals acquire most of their amino acids histidase, urocanase, and fomimino-glutamate hydrolase are from other organisms, thus are lacking much of the amino acid well studied in different organisms by functional and structural synthesis pathways, the amino acid degradation pathways are analysis [9,10] (fomiminoglutamate hydrolase coordinates normally conserved from bacteria to eukaryotes. The study of have only been deposited to PDB recently, with access the amino acid degradation pathways in bacteria will provide ID:1XFK). Imidazolonepropionase is the only one lacking of valuable information in general. The major pathway of structural information in this pathway. In this paper, we histidine degradation in mammals [1,2] and bacteria [3,4] describe the purification and crystallization of the immidazo- involves four enzymes (Fig. 1): (1) histidase (HutH, EC lepropionase (HutI) from Bacillus subtilis. The structure of this 4.3.1.3) deaminates histidine to urocanic acid or urocanate; (2) enzyme will be helpful to deciphering the catalytic mechanism urocanate is then turned into 4(5)-imidazone-5(4)-propionic and provide structural information of this enzyme for all acid (IPA) by urocanase (HutU, EC 4.2.1.49) through organisms. anaerobic addition of a water molecule; (3) then IPA is The hutI gene was amplified by polymerase chain enzymatically split into l-formiminoglutamic acid by imida- reaction (PCR) from genomic DNA of Bacillus subtilis zolonepropionase (HutI, EC 3.5.2.7); (4) the l-formiminoglu- (strain 168) and inserted into pET-21-DEST with an N- tamic acid is then converted to free l-glutamic acid by terminal fusion His6-Tag as described [11]. The correct fomiminoglutamate hydrolase (HutG, EC 3.5.3.8). IPA can insertion was confirmed by DNA sequencing. The recombi- also be non-enzymatically converted to 4-Oxoglutaramic acid nant plasmid was transformed into E. coli BL21 (DE3). [5] and formylisoglutamine [6]. The instability of IPA makes it Transformed cells were cultured in 20 ml Luria–Bertani the last identified intermediate and imidazolonepropionase the (LB) medium containing 100 Ag/ml ampicillin at 310 K last identified enzyme in this pathway [7].InBacillus subtilis, overnight and then inoculated to 1 liter LB medium with the four enzymes cluster in one operon and genetic analysis 100 Ag/ml ampicillin. The culture was grown at 310 K until defines the map order hut(R,P)CHUIG [8]. In this operon, OD600 reached 0.6, the expression of the HutI protein was then induced by addition of isopropyl-h-d-thiogalactoside to * Corresponding author. Tel.: +86 10 62759743; fax: +86 10 62765669. a final concentration of 0.5 mM at 291 K for 20 hours. The E-mail address: [email protected] (X.-D. Su). bacterium cells were harvested by centrifugation at 5000 rpm

154 Y. Yu et al. / Biochimica et Biophysica Acta 1764 (2006) 153–156

Fig. 1. Scheme of histidine degradation pathway. at 277 K for 10 min, and re-suspended in a binding buffer by step during the later purification (Fig. 2B). The first peak (20 mM Tris–HCl, pH 7.5, 500 mM NaCl). After eluted by 75 mM imidazole, 500 mM NaCl, 20 mM Tris– sonication, the lysate which was clarified by centrifugation HCl, pH 7.5 was a mixture of the full-length and degradated and filtration through a 0.22-Am filter, was applied onto a proteins, while the second peak eluted by 250 mM Pharmacia Ni2+-chelate column (GE, USA) previously imidazole, 500 mM NaCl, 20 mM Tris–HCl, pH 7.5 con- equilibrated by the binding buffer. The target protein, tained only the full-length protein. The second peak was containing 421 amino acids, with a molecular mass 45.4 then concentrated and loaded onto a Pharmacia gel filtration kDa, was eluted using a linear gradient of 25 to 500 mM column, Hiload Superdex 75 (GE, USA), for further imidazole in 500 mM NaCl, 20 mM Tris–HCl, pH 7.5 for 5 purification. We deduced from the gel filtration chromato- column volume. There were two not well separated peaks in graphy that it existed as a dimmer in solution. The sample the Ni2+-chelating chromatogram as indicated by two arrows obtained directly from gel filtration was concentrated to 30 (Fig. 2A). The components of the two peaks were checked mg mlÀ1 measured by the Bio-Rad Protein Assay Kit (Bio- by sodium dodecyl sulfate polyacrylamide gel electrophoresis Rad, USA). (SDS-PAGE), and two bands with a molecular mass Crystallization Screen experiments were performed using difference of a few thousand Daltons were shown on the the Hampton Kits, Crystal Screen, Crystal Screen 2 and gel. The separation of the two bands was caused by the N- Index (Hampton Research, USA) by hanging-drop vapor- terminal degradation, as proven later by Western blot diffusion methods at 289 K, and micro-crystals were analysis using anti-His6 antibodies. The full-length protein obtained under many different conditions. The crystals band was detected by the antibodies while the lower growing from the condition of Crystal Screen No. 22 molecular mass band was not (data not shown). In order (30% w/v PEG4000 0.1 M Tris–HCl pH 8.5 0.2 M Sodium to separate the two components, the protein was eluted step Acetate) gave some diffractions, optimization was thus 中国科技论文在线 http://www.paper.edu.cn

Y. Yu et al. / Biochimica et Biophysica Acta 1764 (2006) 153–156 155

Fig. 2. (A) HutI was eluted in a linear gradient. (B) HutI was eluted step by step. See the text for detail.

carried out based on this condition, with varied pH, the degradation was from N-terminus. The purification precipitant and salt concentrations. The Additive Kit strategy was thus further optimized by digesting the N- (Hampton Research, USA) was also used, and benzamidine terminal recombination tag of HutI with thrombin after hydrochloride was found helpful. It was found by accident chelating column purification. The recognizing sequence of that after the protein sample from Nickel chelating column thrombin was underlined and the cutting site was indicated was stored at 277 K for 2–3 days, the protein could form by a arrow within the fusion his-tag sequence: MASMT- crystals with much better diffraction quality after further GGQQMGSSHHHHHHSSGLVPR , GSQSTSLYKKAG- purification by size exclusion chromatography. The crystals SAAAPFT, the HutI sequence then follows. The crystals were checked by SDS-PAGE and proved to contain only with high quality were obtained finally in 20% (w/v) the degraded protein. It was further proved by Western blot PEG4000, 0.1 M Tris–HCl pH 7.5, 0.2 M Sodium Acetate analysis using an anti-His6 antibody (data not shown) that and 2% (w/v) benzamidine hydrochloride with the N- 中国科技论文在线 http://www.paper.edu.cn

156 Y. Yu et al. / Biochimica et Biophysica Acta 1764 (2006) 153–156 terminal cleaved sample (Fig. 3A), whereas the full-length Table 1 protein could only form small crystals with poor diffraction Crystallographic parameters and data processing statistics quality in the same conditions (Fig. 3B). Wavelength (A˚ ) 0.9792 The best X-ray diffraction data were collected on a Resolution (A˚ ) 20–2.0 (2.03–2.0) Completeness (%) 99.8 (99.8) MAR165 CCD detector at beamline BW7A, EMBL outsta- a Rsym (%) 8.2 (22.5) tion, DESY, Hamburg. The crystal was flash frozen and Average I/r(I) 8.3 (3.5) maintained at 100 K by nitrogen gas during data collection. Spacegroup P21 The data were processed with DENZO and SCALEPACK Cell parameters a =57.73 A˚ , b =106.34 A˚ , - [12]. The crystal diffracted to high resolution, and collected to c =66.47 A˚ , b =89.93 ˚ No. of observed reflections 403486 2.0 A in this experiment, and the space group of the crystal No. of independent reflections 54728 was found to be P21, with unit-cell parameters a =57.73 A˚ , Mol/Asym 2 3 À1 b =106.34 A˚ , c =66.47 A˚ , b =89.93-. Assuming two mole- VM (A˚ Da ) 2.25 3 À Solvent content (%) 45 cules per asymmetric unit, it gave a VM value of 2.25 A˚ Da 1 [13], corresponding to a solvent content of 45%. The data (Values in parentheses are for the highest resolution shell.) a ~ ~ collection statistics are listed in Table 1. Rsym = |Iobs ÀIavg|/ Iobs where the summation is over all reflections.

Acknowledgements logy and Development Program of China (863 program 2002BA711A13). Peking University’s 985 and 211 grants are The authors are grateful to Qiang Chen and Tianmin also greatly acknowledged. XDS is a recipient of National Fu for their helpful criticism of the manuscript. This Science Fund for Distinguished Young Scholars of NSFC work was supported by grant from National High Techno- (30325012).

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