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Methylthioribose-1-phosphate isomerase (MtnA) from Bacillus subtilis, the first enzyme in the downstream section of the methionine-salvage pathway, was crystallized using the sitting-drop vapour-diffusion method. Crystals grew using ammonium sulfate as the precipitant at 293 K. They diffracted to 2.5 Å at 100 K using synchrotron radiation and were found to belong to the tetragonal space group P41, with unit-cell parameters a = b = 69.2, c = 154.7 Å. The asymmetric unit contains two molecules of MtnA, with a V M value of 2.4 Å3 Da−1 and a solvent content of 48%.
Methionine is an essential amino acid and plays an important role in a number of cellular functions, including the initiation of protein synthesis, biological methylation of DNA and rRNA and the biosynthesis of cysteine, phospholipids and polyamines. The biosynthesis of polyamines is involved in the control of many biological processes such as carcinogenesis, cell growth and differentiation. In the synthesis of the polymaines spermidine and spermine, methionine is consumed in a 1:1 stoichiometry with formation of the byproduct methylthioadenosine (MTA; Williams-Ashman & Canellakis, 1979 ). Since the amount of methionine in cells is limited and de novo synthesis of methionine is energetically costly, it is essential to salvage methionine and recycle the S atom from MTA (Trackman & Abeles, 1983 ). Moreover, it has long been known that MTA, once converted to adenine and methylthioribose-1-phosphate (MTR-1-P; Ferro et al., 1978 ), is able to regenerate methionine (Williams-Ashman et al., 1982 ; Winans & Bassler, 2002 ). This has been found in a wide variety of organisms, including mammals (Riscoe & Ferro, 1984 ; Riscoe et al., 1988 ), Trypanosoma brucei (Riscoe et al., 1988 ), Saccharomyces cerevisiae (Subhi et al., 2003 ) and Klebsiella pneumoniae (Furfine & Abeles, 1988 ). The overall reaction scheme was determined in K. pneumoniae (Furfine & Abeles, 1988 ). However, the actual enzymes or genes involved in each step have not been unravelled.
A gene-inactivation study revealed that the S-box transcription-termination control system, first identified in Bacillus subtilis, was used for regulation of gene expression in response to methionine availability (Grundy & Henkin, 1998 ). The presence of the S-box motif provided the first indication that the mtnKA and mtnWXBD genes could play a role in recycling MTA (Murphy et al., 2002 ). In 2003, we succeeded in showing the exact nature of five enzymes (MtnA, MtnW, MtnX, MtnB, MtnD) from B. subtilis in the six steps of the downstream section of the methionine-salvage pathway (Ashida et al., 2003 ; Balakrishnan et al., 1993 ; Sekowska et al., 2004 ). The first step of the pathway in B. subtilis is catalyzed by MtnA, which converts MTR-1-P into methylthioribulose-1-phosphate (MTRu-1-P).
The first ubiquitous enzyme of the pathway, MtnA, belongs to a family of proteins related to the α-subunit of eukaryotic translation initiation factor 2B (eIF2B), which is a heteropentameric protein composed of α– subunits. Recently, the crystal structures of MtnA-homologous proteins [Ypr118w from S. cerevisiae and an archaeal regulatory subunit (aIF2Bα) from Pyrococcus horikoshii OT3] have been reported (Bumann et al., 2004 ; Kakuta et al., 2004 ). Despite structural analyses and genetic studies, the active site remains unclear. The primary structure and molecular weight of MtnA from B. subtilis were very different from those of Ypr118w (38% identity, 45 kDa) and aIF2Bα (32% identity, 31 kDa). We have also established the generation of MTR-1-P, the substrate of MtnA, using recombinant B. subtilis MtnK. For the purpose of investigation into the detailed catalysis of MtnA, the crystallization of MtnA complexed with MTR-1-P is currently in progress.
The methionine-salvage pathway is likely to be compartmentalized because of the extreme reactivity of the S atom towards dioxygen and radicals. Furthermore, the structure–function relationship of the series of B. subtilis enzymes remains unclear and their three-dimensional structures are unknown. Therefore, structural comparison between MtnA and its homologues will not only provide an evolutionary insight into two different catalytic mechanisms, but the three-dimensional structure of MtnA will also become an important first step in understanding the whole protein–protein network in the pathway. We report here the crystallization and the preliminary X-ray crystallographic study of MtnA from B. subtilis.
The full-length mtnA gene was amplified from genomic DNA by PCR, which was performed with the following forward and reverse primers: 5′-GGAATTCCATATGACCCATTCATTTGCTG-3′ and 5′-CGGGATCCAAATGAGCAAAGTCC-3′. The products of PCR were digested with NdeI and BamHI. The fragments were ligated at the NdeI/BamHI site of pET15b (Novagen).
Escherichia coli strain BL21 (DE3) cells harbouring the mtnA expression plasmid were grown for 8 h in LB medium containing 50 µg ml−1 ampicillin at 310 K. The cells were grown for a further 16 h at 303 K. The cells were then harvested by centrifugation at 6000g for 30 min. The cell pellet was resuspended in buffer A (50 mM Na HEPES pH 7.4, 500 mM NaCl and 10 mM imidazole) and 1 mM PMSF and then disrupted using a French press.
The disrupted cells were centrifuged at 400 000g at 277 K for 30 min. The supernatant was loaded onto a 5 ml HiTrap Chelating column (Amersham Pharmacia Biotech) equilibrated with buffer A. The unbound proteins were flushed with buffer A. MtnA with an N-terminal histidine tag was eluted with a 150–225 mM imidazole gradient. Further purification to MtnA was carried out using a HiLoad 26/60 Superdex 75 prep-grade column (Amersham Pharmacia Biotech) equilibrated with 50 mM Na HEPES pH 7.4 containing 1 mM EDTA. Pooled fractions were concentrated to 30 mg ml−1 with a Vivaspin 20 ml (5000 MWCO PES, Vivascience) and used for crystallization without removal of the N-terminal histidine tag.
Crystallization screening using 30 mg ml−1 MtnA (in 50 mM Na HEPES pH 7.4 and 1 mM EDTA) was initially performed with Crystal Screen and Crystal Screen 2 (Hampton Research) in 96-well sitting-drop plates (Corning) at 293 K. The drop size was 2 µl, with a protein:reservoir ratio of 1:1. Oval-shaped crystals with maximum dimensions of 0.15 × 0.05 × 0.02 mm were obtained using Crystal Screen 2 condition No. 23 [100 mM MES pH 6.5, 1.6 M ammonium sulfate, 10%(v/v) 1,4-dioxane] within two weeks (Fig. 1 a). However, the crystal diffracted with high mosaicity (1.6). Another small crystal appeared using Crystal Screen 2 condition No. 42 [100 mM Tris pH 8.5, 1.5 M ammonium sulfate, 12%(v/v) glycerol] after a month. To refine the conditions, we varied the pH, buffer and concentrations of protein or precipitant and attempted the hanging-drop vapour-diffusion method using a 2 µl drop containing equal volumes of protein and reservoir solution suspended over 400 µl reservoir solution. Optimized crystallization was performed with the sitting-drop vapour-diffusion method at 293 K with a drop (2 µl) containing equal volumes of protein solution (10 mg ml−1 MtnA) and reservoir solution [50 mM MES, 50 mM Tris (final pH 7.4), 1.55 M ammonium sulfate, 5%(v/v) 1,4-dioxane, 6%(v/v) glycerol] suspended over 100 µl reservoir solution. Crystals suitable for diffraction with maximum dimensions 0.1 × 0.05 × 0.03 mm appeared after one week (Fig. 1 b).
The crystals were soaked for a few seconds in reservoir solution containing 20%(v/v) glycerol and 2.0 M ammonium sulfate. Flash-cooled crystals were then mounted in the nitrogen stream at 100 K. X-ray diffraction data were collected to 2.5 Å on a Rigaku R-AXIS V detector using synchrotron radiation of wavelength 1.0 Å at the BL38B1 beamline at SPring-8 (Fig. 2 ). Data collection was performed with a total oscillation range of 180°, a step size of 1.0° and an exposure time of 100 s. All diffraction data were processed using the HKL2000 program package (Otwinowski & Minor, 1997 ) and the CCP4 program suite (Collaborative Computational Project, Number 4, 1994 ).
Form II crystals diffracted well to 2.5 Å at 100 K using the BL38B1 beamline at SPring-8 and were found to belong to the tetragonal space group P41 (or P43), with unit-cell parameters a = b = 69.2, c = 154.7 Å. The value of the Matthews coefficient is 2.4 Å3 Da−1 for a dimer in the asymmetric unit, corresponding to a solvent content of 48%, a typical value for protein crystals (Matthews, 1968 ). This is corroborated by analysis of the self-rotation function at χ = 180° (POLARRFN; Collaborative Computational Project, Number 4, 1994 ; Fig. 3 ). The result indicates the existence of a local twofold axis at (θ, ϕ, χ) = (90, 30, 180°). The peak intensity with respect to the crystallographic axis was 41.8%. Details of data processing and statistics are shown in Table 1 . Structure determination was performed by molecular replacement using MOLREP v.7.3 (Vagin & Teplyakov, 1997 ) from the CCP4 v.4.2 software suite (Collaborative Computational Project, Number 4, 1994 ), using the coordinates of Ypr118w from S. cerevisiae (PDB code http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1w2w) as a search model. The results of molecular replacement suggest that the crystals contain two monomers per asymmetric unit and belong to space group P41. Refinement of structural details is now in progress.
We are grateful to Drs K. Hasegawa and H. Sakai for their kind help in data collection at BL38B1 at SPring-8 and to Drs A. Nakagawa and E. Yamashita, Institute for Protein Research, Osaka University for their help in initial X-ray diffraction spot measurement at BL44XU at SPring-8. The synchrotron-radiation experiments were performed at SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI; 2001A0336-CL-np) and the Institute for Protein Research, Osaka University (C04A44XU-7119-N). This research was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, partly by the PRESTO project, Japan Science and Technology Agency and the Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.