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Prokaryotic 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MtaN) is a multifunctional enzyme that can hydrolyze S-adenosyl-l-homocysteine (SAH) and S-methyl-5′-thioadenosine (MTA) to give S-ribosyl-l-homocysteine (SRH) and S-methyl-5′-thioribose (MTR), respectively. This reaction plays a key role in several metabolic pathways, including biological methylation, polyamine biosynthesis, methionine recycling and bacterial quorum sensing. Structurally, MtaN belongs to the MtnN subfamily of the purine nucleoside phosphorylase (PNP)/uridine phosphorylase (UDP) phosphorylase family. Aeromonas hydrophila has two MtnN subfamily proteins: MtaN-1, a periplasmic protein with an N-terminal signal sequence, and MtaN-2, a cytosolic protein. In this study, MtaN-1 from Aeromonas hydrophila was successfully expressed and purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. Crystals of the protein in complex with the substrate SAH were obtained and diffracted to a resolution of 1.4 Å. The crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 102.7, c = 118.8 Å. The asymmetric unit contained two molecules of MtaN-1 complexed with SAH.
S-Adenosylmethionine (SAM) is an important nucleoside that serves as an activated-group donor in diverse metabolic and biosynthetic reactions mediated by radical SAM enzymes (Parveen & Cornell, 2011 ). Radical SAM enzymes reductively cleave SAM to generate the highly reactive 5′-deoxyadenosyl radical and catalyze the ensuing reactions by utilizing this radical. During this process, highly reactive product inhibitors of these reactions, such as 5′-methylthioadenosine (MTA), S-adenosylhomocysteine (SAH) and 5′-deoxyadenosine (5′-DOA), can accumulate, resulting in enzyme inhibition (Choi-Rhee & Cronan, 2005 ; Frey & Magnusson, 2003 ).
In bacteria, the MtnN subfamily protein MTA/SAH nucleosidase (MtaN) is known as a major source of metabolism of the SAH, MTA and 5′-DOA generated by various SAM-utilization pathways (Parveen & Cornell, 2011 ; Longshaw et al., 2010 ). This enzyme can irreversibly hydrolyze the glycosidic bond of MTA, SAH and 5′-DOA to give S-methyl-5′-thioribose (MTR), S-ribosyl-l-homocysteine (SRH) and 5′-deoxyribose, respectively (Duerre, 1962 ). Thus, inhibition of MtaN activity could cause an accumulation of MTA and SAH within bacterial cells and thus also inhibit radical SAM enzymes, such as methylases and polyamine synthases, which are essential for bacterial growth (Beeston & Surette, 2002 ). Various antibacterial drugs that target MtaN have been developed to date, although they appear to have limited efficacy (Longshaw et al., 2010 ). Thus, an understanding of the underlying mechanisms of MtaN is necessary for the development of new and effective antibacterial treatments.
The Gram-negative, rod-shaped bacterium Aeromonas hydrophila is an opportunistic pathogen that infects humans, fish and other lower vertebrates and is found in stagnant and flowing fresh water at the interface of sea water and fresh water (Park et al., 2011 ). This pathogen greatly affects both human health and animal breeding. Previous studies have found that A. hydrophila can cause the following four categories of infection: cellulitis, acute diarrhoeal disease, sepsis and other infections (Davis et al., 1978 ; Janda & Abbott, 2010 ). A. hydrophila has two MtnN subfamily proteins: MtaN-1 and MtaN-2. MtaN-1 appears to be a periplasmic protein because of the presence of a predicted N-terminal signal sequence for secretion, whereas MtaN-2 is a cytosolic protein owing to the lack of a predicted signal sequence. The presence of periplasmic MtaN-1 is interesting because MtaN should function in the cytosol to metabolize radical products.
A total of 11 bacterial and plant MtaN structures have been solved (Lee et al., 2001 , 2003 , 2005 ; Siu et al., 2008 ) using X-ray crystallography and are available in the Protein Data Bank. However, the crystal structure, molecular function and mechanism of action of the MtaN proteins from A. hydrophila remain largely uncharacterized. Moreover, no previous studies have investigated the structure and function of periplasmic MtaN. We reasoned that establishing the three-dimensional structure of MtaN-1 from A. hydrophila could possibly provide an understanding of the function of the A. hydrophila periplasmic MtaN. Here, we report the purification, crystallization and preliminary X-ray analysis of the intact form of MtaN-1 from A. hydrophila.
To construct a plasmid expressing A. hydrophila MtaN-1 (residues 31–278, lacking the signal sequence), we amplified DNA fragments encoding A. hydrophila MtaN-1 (residues 31–278) from the genomic DNA of an A. hydrophila strain by PCR using standard methods. The PCR primers were constructed in such a way that cloning of the PCR product into the EcoRI and HindIII sites of the pPROEX-HTA vector (Invitrogen, USA) would generate a protein containing a hexahistidine tag (MSYYHHHHHH), a spacer region (DYDIPTT) and a TEV protease cleavage site (ENLYFQ) at the N-terminus. The primers were MtaN-1_his-6_forward, 5′-CCGGAATTCATGGCGGCCAAGCCG-3′, and MtaN-1_his-6_reverse, 5′-CCCAAGCTTTCAGGCTTTGTTGAAGCCGT-3′, and the resulting plasmid was named pPROTEXHTA-MtaN-1.
Escherichia coli BL21 (DE3) cells were transformed with the pPROTEXHTA-MtaN-1 construct for overexpression. To produce a sufficient amount of protein for structural studies, cells were grown in 1 l Luria–Bertani (LB) broth with 50 µg ml−1 ampicillin at 310 K until an OD600 of 0.7–0.9 was reached. Protein overexpression was then induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside to the culture at 303 K. The cells were harvested by centrifugation 7 h after induction and were stored at 193 K until use.
The harvested cell pellets were defrosted, suspended in lysis buffer consisting of 20 mM Tris pH 8.0, 150 mM NaCl, 2 mM β-mercaptoethanol and disrupted by sonication. The cell debris was removed by centrifugation at 45 000g for 30 min at 273 K. The resulting supernatant fraction was mixed with Ni–NTA affinity resin (GE Healthcare, USA) that had been pre-incubated with Tris buffer and the mixture was mixed for 30 min at 4°C. The resin was washed with lysis buffer supplemented with 20 mM imidazole. The target protein with the hexahistidine tag was eluted with 30 ml of lysis buffer including 250 mM imidazole. The eluted fractions were analyzed by SDS–PAGE. The fractions containing MtaN-1 were pooled and β-mercaptoethanol was added to a final concentration of 10 mM. This solution was incubated with recombinant TEV protease overnight at 298 K to remove the hexahistidine tag. The reaction mixture was diluted fourfold with 20 mM Tris pH 8.0 buffer and then loaded onto a Q anion-exchange column (HiTrap Q; GE Healthcare, USA) for further purification. The protein was eluted from the column by a salt gradient to 1 M NaCl in 20 mM Tris pH 8.0. The collected fractions containing the MtaN-1 protein were pooled, concentrated using Centriprep concentrators (Millipore, USA) and separated on a HiLoad Superdex 200 gel-filtration column (GE Healthcare, USA) that had been pre-equilibrated with lysis buffer. The purity of the final protein was confirmed by 15% SDS–PAGE and Coomassie Blue staining. The purified protein was stored at 277 K for use within a week or stored frozen at 193 K until use.
Prior to crystallization, the pooled sample of purified MtaN-1 was concentrated to 10 mg ml−1 using a Vivaspin centrifugal concentrator fitted with a 10 kDa molecular-weight cutoff filter (Millipore, USA). The crystallization conditions were initially screened using Crystal Screen HT, a high-throughput sparse-matrix screening kit (Hampton Research, USA). Crystals were grown by the sitting-drop microbatch method at various temperatures (279, 288 and 295 K). Crystals of the recombinant MtaN-1 protein were obtained by vapour diffusion only at 288 K. To obtain a crystal of MtaN-1 in complex with SAH, the crystal of MtaN-1 was gently removed from the crystallization drop and briefly incubated in a solution consisting of 6%(w/v) polyethylene glycol 4000, 0.1 M sodium acetate trihydrate pH 4.6, 0.5 mM SAH.
For data collection under cryogenic conditions, crystals were removed from the drop, soaked for 3–5 s in a 10 µl droplet of cryoprotectant solution prepared by adding 25%(v/v) glycerol to the optimized crystallization conditions and then flash-cooled in a liquid-nitrogen stream. For the crystal of MtaN-1 complexed with SAH, we collected X-ray diffraction data at 100 K at a wavelength of 1.0000 Å using an ADSC Q310 CCD detector on beamline 5C of Pohang Light Source (PLS), Republic of Korea. A total of 180 diffraction images were recorded from a single crystal. The complete diffraction data sets were subsequently processed, merged and scaled with HKL-2000 (Otwinowski & Minor, 1997 ). The data-collection statistics are given in Table 1 .
Recombinant MtaN-1 was overexpressed and purified with a yield of 12 mg from 1 l LB medium culture using the protein-purification methods described in §2. The purity of the protein sample was >96% as judged by Coomassie-stained SDS–PAGE. The protein sample was successfully crystallized under several conditions from the initial crystallization screening trials by the sitting-drop vapour-diffusion method. Subsequent refinement of the initial conditions resulted in the production of larger-sized crystals of 0.07 × 0.07 × 0.03 mm (Fig. 1 ) in 2 d using droplets consisting of 1 µl protein solution (10 mg ml−1 protein in 20 mM Tris buffer pH 8.0 containing 150 mM NaCl) and 1 µl reservoir solution consisting of 6%(w/v) polyethylene glycol 4000, 0.1 M sodium acetate trihydrate pH 4.6.
We collected diffraction data to 1.4 Å resolution for the complex of MtaN-1 with SAH (Fig. 2 ). Based on the diffraction data, the crystal belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 102.6, c = 118.7 Å. The asymmetric unit contains two molecules of MtaN-1 complexed with SAH. Analysis of the diffraction along the h, k and l axes clearly demonstrates that the c axis of the crystal is a 31 or 32 axis. The diffraction data set had a resolution range of 49.5–1.4 Å with 100% completeness and an R merge of 6.4%. Data-collection statistics are given in Table 1 . We attempted to determine the structure of A. hydrophila MtaN-1 complexed with SAH by the molecular-replacement method with MOLREP (Vagin & Teplyakov, 2010 ) by utilizing the structure of Helicobacter pylori MtaN (9.4% sequence identity; PDB entry 3nm4; Ronning et al., 2010 ) as a search model. This approach produced a clear solution. Cross-rotation and translation-function calculations confirmed that two molecules are present in the asymmetric unit; the corresponding Matthews coefficient (Matthews, 1968 ) and solvent content are 3.25 Å3 Da−1 and 62.2%, respectively. The initial model of MtaN-1 built was refined automatically with PHENIX (Adams et al., 2010 ; Fig. 3 ). The results showed that the crystals obtained were a complex of MtaN-1 and SAH. Currently, we are in the process of building a model and refining the structure of the MtaN-1 complex with SAH together with its interpretation.
This work was supported by the National Natural Science Foundation of China (Grant No. 31200556 to YX and Grant No. 21272031 to C-SQ), the China Postdoctoral Science Foundation (Grant No. 2013M540229 to YX) and the Fundamental Research Funds for the Central Universities (Grant No. DC13010308 to YX). We gratefully acknowledge access to beamline 5C at Pohang Light Source (PLS), Republic of Korea. We are grateful to Inseong Jo at Seoul National University for helpful advice during data collection.