We report a 2. The two moieties are produced by two

We report a 2. The two moieties are produced by two separate biosynthetic processes which are then covalently linked to yield thiamin phosphate [1 2 This process is well BC 11 hydrobromide studied in prokaryotes but is still poorly understood in eukaryotes. Thiamin synthesis has been studied to some degree in yeast; in the gene product THi5 is responsible for the synthesis of 4-amino-5-(hydroxymethyl)-2-methylpyrimidine phosphate in yeast [3–5]. THi5 appears to be conserved in eukaryotes with thiamin biosynthetic pathways [3–5]. THi5 belongs to a large superfamily known as the NMT1/THI5-like domain proteins (PFam entry PF09084 comprising 7 204 sequences). However the majority of members of the NMT1/THI5-like superfamily are found in eubacteria especially (4 295 sequences in 1 354 species). While there is some structural information for the superfamily—for example a homolog in RB50 containing pyrimidine/thiamin biosynthesis precursor-like domain BC 11 hydrobromide which shed new light on potential proteins taking part in thiamin biosynthesis in this organism. Materials and methods Cloning expression and purification Selenomethionine (Se-Met) substituted “type”:”entrez-protein” attrs :”text”:”CAE31940″ term_id :”33568027″ term_text :”CAE31940″CAE31940 protein was produced using standard MSCG protocols as described by Zhang et al. [6]. Briefly gene BB1442 from RB50 BC 11 hydrobromide was cloned into a p15TV LIC plasmid using ligation independent cloning [7–9]. The gene was overexpressed in BL21-CodonPlus(DE3)-RIPL cells in Se-Met-containing LB media at 37.0 °C until the optical density at 600 nm reached 1.2. The cells were induced by isopropyl-β-D-1-thiogalactopyranoside incubated at 20 then.0 °C overnight and pelleted by centrifugation. Harvested cells were sonicated in lysis buffer (300 mM NaCl 50 mM HEPES pH 7.5 5 % glycerol and 5 mM imidazole) the lysed cells were spun down for 15 min at 16 0 RPM and the supernatant was applied to a nickel chelate affinity resin (Ni–NTA Qiagen). The resin was washed with wash buffer (300 mM NaCl 50 mM HEPES pH 7.5 5 % glycerol and 30 mM imidazole) and the protein was eluted using Itga1 elution buffer (300 mM NaCl 50 mM HEPES pH 7.5 5 % glycerol and 250 mM imidazole). The N-terminal polyhistidine tag (His-Tag) was removed by digestion with recombinant TEV protease and the digested protein was passed through a second affinity column. The flow through was dialyzed against a solution containing 300 mM NaCl 10 mM HEPES pH 7.5 and 1 mMTCEP. Purified protein was concentrated to 36 flash-frozen and mg/mL in liquid nitrogen. Crystallization Crystals of “type”:”entrez-protein” attrs :”text”:”CAE31940″ term_id :”33568027″ term_text :”CAE31940″CAE31940 used for data collection were grown by the sitting drop vapor diffusion method. The well solution consisted of 0.2 M ammonium acetate 30 % w/v PEG4000 and 0.1 M tri-sodium citrate at pH 5.6. Crystals were grown at 293 K and formed after 1 week of incubation. Immediately after harvesting crystals were transferred into cryoprotectant solution (Paratone-N) BC 11 hydrobromide without mother liquor washed twice in the solution and flash cooled in liquid nitrogen. Data collection and processing Data were collected at 100 K at the 19-ID beamline (ADSC Q315 detector) of the Structural Biology Center BC 11 hydrobromide [10] at the Advanced Photon Source (Argonne National Laboratory Argonne Illinois USA). The beamline was controlled by HKL-3 0 [11]. Diffraction data were processed with HKL-2 0 [11]. Data collection structure refinement and determination BC 11 hydrobromide statistics are summarized in Table 1. Table 1 Crystallographic parameters and data collection and refinement statistics Structure solution and refinement The structure of the Se-Met-substituted protein was solved using single-wavelength anomalous diffraction (SAD) and an initial model was built with HKL-3000. HKL-3000 is integrated with SHELXC/D/E [12] MLPHARE DM ARP/wARP CCP4 [13] RESOLVE and SOLVE [14]. The resulting model was further refined with REFMAC5 [15] and COOT [16]. MOLPROBITY ADIT and [17] [18] were used for structure validation. The coordinates and experimental structure factors were deposited to PDB with accession code 3QSL. Bioinformatics analyses Sequence homology searches were performed with PSI-BLAST [19] and structural homology searches were done with HHpred [20 21 with amino acid sequence of {“type”:”entrez-protein” attrs :{“text”:”CAE31940″ term_id :”33568027″ term_text.