Structural Analysis of the NH<sub>3</sub>-dependent NAD<sup>+</sup> Synthetase from Deinococcus radiodurans
Structural Analysis of the NH3-dependent NAD+ Synthetase from Deinococcus radiodurans
Bulletin of the Korean Chemical Society. 2014. Sep, 35(9): 2863-2866
Copyright © 2014, Korea Chemical Society
  • Received : April 03, 2014
  • Accepted : May 29, 2014
  • Published : September 20, 2014
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About the Authors
Young Woo Park
Hyun Ku Yeo
Jae Young Lee

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Materials and Methods
Protein Expression and Purification . The nadE gene encoding NADS was amplified by polymerase chain reac reaction using genomic DNA of D. radiodurans as a template. It was inserted into the NdeI/BamHI-digested expression vector pET-28b(+) (Novagen), resulting in a twenty-residue hexahistidine-containing tag at its N-terminus. The recombinant dra NADS was expressed in E. coli BL21 (DE3) star pLysS cells (Invitrogen). Overexpression of the recombinant protein was induced with 1.0 mM isopropyl β-D-thiogalacto-pyranoside (IPTG) and the cells were continuously cultured at 303 K for 4 h. After harvest of the cells by centrifugation at 4200 g for 10 minutes at 277 K, the pellet was resuspended in a lysis buffer [20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride] and homogenized by an ultrasonic processor. The insoluble fraction was removed by centrifugation at 31000 g for 60 minutes at 277 K and the recombinant dra NADS in the supernatant fraction was purified by three chromatographic steps. The first step was a metal-chelate chromatography on Ni-NTA resin (GE Healthcare). The His-tagged dra NADS protein was eluted with buffer A (20 mM Tris- HCl at pH 8.0, 0.5 M NaCl, 10% glycerol) containing 300 mM imidazole followed by enzymatic removal of the Histag by overnight incubation with prescission protease. The uncleaved His-tagged protein and prescission protease were removed from the target dra NADS applying to Ni-NTA affinity column. The next step was gel filtration on a Superdex-75 column (GE Healthcare), employing an elution buffer of 0.2 M NaCl, 20 mM Tris-HCl at pH 8.0, 1 mM DTT, 5 mM MgCl 2 and 5% glycerol. The purified protein was concentrated to 30 mg/mL using Centricon YM-10 (Millipore) and aliquots of the protein were stored at 193 K.
Crystallization and Data Collection . Initial crystallization was performed by the sitting-drop vapor diffusion method using 96-well CrystalQuick plates (Greiner Bio- One) and commercial screens (Hampton Research; Qiagen; Emerald Biosystems) at 296 K. Crystals of the dra NADS were initially grown in several conditions containing polyethylene glycol (PEG) 4000 and lithium sulfate, which were further optimized to 20% PEG 4000, 0.2 M lithium sulfate, 0.1 M MES at pH 6.0. The crystals grew reproducibly up to approximately 0.1 × 0.2 × 0.05 mm within 3 days. Crystals were transferred into a cryoprotectant solution containing 20% glycerol in reservoir solution and then flash-cooled in liquid nitrogen. X-ray diffraction data were collected to 2.6 Å at 100 K using an ADSC Quantum 210 CCD image-plate detector on Beamline SB-I of the Pohang Accelerator Laboratory, Korea. The crystals belonged to the orthorhombic space group P 2 1 2 1 2 1 , with unit cell parameters of a = 113.23 Å, b = 114.15 Å, and c = 121.62 Å. Two dra NADS dimers were present in an asymmetric unit, giving a solvent fraction of 61.1%. All data were processed and scaled with iMOSFLM program suite. 4,10
Structure Determination and Refinement . The dra NADS crystal structure was solved by molecular replacement using program PHASER 11 using the ban NADS coordinates (PDB code 2PZ8) 7 as a search model. Subsequently, the initial model was further improved by the alternating cycles of manual building using the COOT program 12 and the model was refined with the PHENIX program. 13 The refined model was evaluated using MolProbity. 14 X-ray data collection and refinement statistics are presented in Table 1 . The coordinate and structure factor have been deposited in the Protein Data Bank under accession number 4Q16.
Data collection and refinement statistics
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aRmerge = Σh Σi| I (h,i) − < I (h)> | /Σh ΣiI (h,i), where I (h,i) is the intensity of the i th measurement of reflection h and < I (h) is the mean value of I (h,i) for all i measurements. bRwork = Σ||Fobs| − |Fcalc|| / Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively. Rfree was calculated as Rwork using a randomly selected subset of ~5% of the unique reflections not used for structure refinement. Values in parentheses refer to the highest resolution shell.
Result and Discussion
The crystal structure of dra NADS was determined by molecular replacement at 2.60 Å resolution. The structure was refined to crystallographic R work and R free values of 23.9% and 29.2%, respectively. The refined model (PDB code 4Q16) contained 1,064 residues of the two independent dra NADS dimers, 8 molecules of sulfate ion, and 362 water molecules in the asymmetric unit. In each subunit, an internal region of the polypeptide chain (Glu221–Arg224 in subunit A, Val215–Glu221 in subunit B, Arg213–Pro228 in subunit C, and Asp219–Asp222 in subunit D) and terminal residues (Met1–Pro7 and Gly282–Ser287 in subunit A, Met1–Leu6 and Gly282–Ser287 in subunit B, Met1–Pro10 and Gly282–Ser287 in subunit C, and Met1–Pro7 and Glu283–Ser287 in subunit D) were disordered. Each dra NADS subunit has single α/β core domain consisting of 4 parallel β-stands, 11 α-helices and connecting loops, which shows typical Rossman fold. The core domains of the two subunits are formed homodimer, with approximate dimensions of 42 Å × 53 Å × 65 Å (Fig. 1(b) ). The solvent accessible surface area buried at the interface in the dimeric unit is about 2556 Å2 (19% of their individual accessible surface areas). The extended C-terminal loops (residues 266-276) and the α-helix bundle (residues 116-159) composed of two helices mainly participate in dimerization ( Fig. 1(c) ). Especially, the C-terminal segment and helix-loop motif (residues 116-136) are similar to arms of clamp and anchor the subunits to each other ( Fig. 1(d) ). The dimeric interface is predominantly contributed by both hydrophobic interactions and hydrogen bonds.
The structural comparisons showed that the dra NADS structure is highly similar with other NH 3 -depenent NADS structures, (i) bsu NADS (PDB code 2NSY, r.m.s. deviation of 1.0 Å for 267 equivalent Cα positions in residues 11-280 of dra NADS, a Z-score of 39.8, and a sequence identity of 58%), 5 (ii) ban NADS (PDB code 2PZ8, r.m.s. deviation of 1.1 Å for 269 equivalent Cα positions in residues 10-281 of dra NADS, a Z-score of 39.6, and a sequence identity of 59%), 7 (iii) eco NADS (PDB code 1WXI, r.m.s. deviation of 1.1 Å for 258 equivalent Cα positions in residues 11-281 of dra NADS, a Z-score of 36.7, and a sequence identity of 59%), 6 and (iv) ftu NADS (PDB code 3FIU, r.m.s. deviation of 1.6 Å for 234 equivalent Cα positions in residues 25-272 of dra NADS, a Z-score of 30.0, and a sequence identity of 36%) 9 ( Fig. 2(a) ).
Although the general NH 3 -depenent NADS proteins from bacterial species have a substrate preference for NaAD, the ftu NADS mainly catalyzes from nicotinic acid mononucleotide (NaMN) to nicotinamide mononucleotide (NMN) different from general NADS enzymes. Multiple sequence alignment and structural superposition results showed that the substrate binding site of the apo dra NADS structure is well accommodate with NaAD. When the apo dra NADS structure is superimposed with bsu NADS-NaAD complex structure, the conformation of its residues involved in substrates binding site is almost identical with bsu NADS-NaAD complex structure ( Fig. 2(b) ). However, the conformation of ftu NADS substrate binding site is a little different. The Tyr41, Gly157, Leu260 and Asn263 residues in the dra NADS located at the NaAD binding pocket were replaced with Ser23, Gln133, Trp233 and Arg236 in ftu NADS. In particular, the conserved His266 residue which enabled to bind NaAD in dra NADS was oriented in the opposite direction in ftu NADS structure. The orientation of the His266 makes it possible for the NaAD to access into the binding pocket because the residue can avoid a crash with the adenosyl group of NaAD. While the corresponding residue His239 in ftu NADS was stabilized by a hydrogen bond and a stacking interaction with Tyr27, the same orientation of His266 would be blocked by the side chain of Trp278 which is missing in ftu NADS but highly conserved in other NADS proteins ( Fig. 2 ).
In summary, we determined the NH 3 -dependent dra NADS in apo form and analyzed the substrate binding sites of NaAD and ATP. The structure revealed a compact homo-dimer with an extended dimeric interface mediated by mainly the two pairs of helices α5 and α6. Similarities in the overall structure and the location of the substrate binding site residues of dra NADS to other NADS proteins suggest that the dra NADS seems to prefer NaAD as a substrate to synthesize NAD + . In addition, our structural data can provide an idea related in ammonia detection through further research on biochemical mechanism of the dra NADS.
We thank the staff members at Pohang Accelerator Laboratory Beamline 7A SB-I for their help with data collection. This work was supported by the Agriculture Research Center (ARC, 710003-03) program of the Ministry for Food, Agriculture, Forestry and Fisheries, Korea.
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