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Identification, Characterization, and Crystal Structure ofBacillus subtilis Nicotinic Acid Mononucleotide Adenylyltransferase

2002; Elsevier BV; Volume: 277; Issue: 5 Linguagem: Inglês

10.1074/jbc.m109670200

1083-351X

Andrea Olland, Kathryn Underwood, Robert Czerwiński, Mei-Chu Lo, Ann Aulabaugh, Joel Bard, Mark Stahl, W.S. Somers, Francis X. Sullivan, Rajiv Chopra,

Enzyme Structure and Function

The nadD gene, encoding the enzyme nicotinic acid mononucleotide (NaMN) adenylyltransferase (AT), is essential for the synthesis of NAD and subsequent viability of the cell. The nadD gene in Bacillus subtilis(yqeJ) was identified by sequence homology with other bacterial nadD genes and by biochemical characterization of the gene product. NaMN AT catalyzes the reversible adenylation of both NaMN and the nicotinamide mononucleotide (NMN) but shows specificity for the nicotinate. In contrast to other known NMN ATs, biophysical characterizations reveal it to be a dimer. The NaMN AT crystal structure was determined for both the apo enzyme and product-bound form, to 2.1 and 3.2 Å, respectively. The structures reveal a "functional" dimer conserved in both crystal forms and a monomer fold common to members of the nucleotidyl-transferase α/β phosphodiesterase superfamily. A structural comparison with family members suggests a new conserved motif (SXXXX(R/K)) at the N terminus of an α-helix, which is not part of the shared fold. Interactions of the nicotinic acid with backbone atoms indicate the structural basis for specificity. The nadD gene, encoding the enzyme nicotinic acid mononucleotide (NaMN) adenylyltransferase (AT), is essential for the synthesis of NAD and subsequent viability of the cell. The nadD gene in Bacillus subtilis(yqeJ) was identified by sequence homology with other bacterial nadD genes and by biochemical characterization of the gene product. NaMN AT catalyzes the reversible adenylation of both NaMN and the nicotinamide mononucleotide (NMN) but shows specificity for the nicotinate. In contrast to other known NMN ATs, biophysical characterizations reveal it to be a dimer. The NaMN AT crystal structure was determined for both the apo enzyme and product-bound form, to 2.1 and 3.2 Å, respectively. The structures reveal a "functional" dimer conserved in both crystal forms and a monomer fold common to members of the nucleotidyl-transferase α/β phosphodiesterase superfamily. A structural comparison with family members suggests a new conserved motif (SXXXX(R/K)) at the N terminus of an α-helix, which is not part of the shared fold. Interactions of the nicotinic acid with backbone atoms indicate the structural basis for specificity. nicotinic acid mononucleotide adenylyltransferase(s) nicotinic acid adenine dinucleotide nicotinamide mononucleotide high pressure liquid chromatography multiple wavelength anomalous dispersion phosphopantetheine adenylyltransferase glycerol-3-phosphate cytidylyltransferase mononucleotide adenylyltransferase from M. jannaschii mononucleotide adenylyltransferase from M. thermoautotrophicum NAD is an essential molecule in all living cells. In addition to its role in oxidation reduction reactions, in which NAD(H) and its phosphorylated form, NADP(H), act as hydride donors and acceptors, NAD is also important for other cellular processes, such as the activity of NAD-dependent DNA ligases, mono- and poly(A)DP-ribosylation of proteins, and production of the intracellular calcium-mobilizing molecules cADPR and NaADP (1Ziegler M. Eur. J. Biochem. 2000; 267: 1550-1564Crossref PubMed Scopus (244) Google Scholar,2Petit M.A. Ehrlich S.D. Nucleic Acids Res. 2000; 28: 4642-4648Crossref PubMed Scopus (46) Google Scholar). NAD is synthesized via a multi-step de novo pathway or via a pyridine salvage pathway. The enzyme nicotinic acid mononucleotide (NaMN)1 adenylyltransferase (AT, EC 2.7.7.18) sits at the convergence of these two pathways. NaMN AT catalyzes the conversion of ATP and NaMN to nicotinic acid adenine dinucleotide (NaAD) (Fig. 1) that is directly processed to NAD by NAD synthetase. The nadD gene, encoding NaMN AT, was the first enzyme demonstrated to be essential for NAD biosynthesis and bacterial cell survival by both the de novo and salvage pathways (3Hughes K.T. Ladika D. Roth J.R. Olivera B.M. J. Bacteriol. 1983; 155: 213-221Crossref PubMed Google Scholar). A number of enzymes demonstratingin vitro adenylyltransferase activity for NaMN and NMN have been identified in eukarya, archaea, and bacteria (4Magni G. Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 135-182PubMed Google Scholar, 5Balducci E. Orsomando G. Polzonetti V. Vita A. Emanuelli M. Raffaelli N. Ruggieri S. Magni G. Natalini P. Biochem. J. 1995; 310: 395-400Crossref PubMed Scopus (27) Google Scholar, 6Raffaelli N. Emanuelli M. Pisani F.M. Amici A. Lorenzi T. Ruggieri S. Magni G. Mol. Cell Biochem. 1999; 193: 99-102Crossref PubMed Google Scholar, 7Natalini P. Ruggieri S. Raffaelli N. Magni G. Biochemistry. 1986; 25: 3725-3729Crossref PubMed Scopus (41) Google Scholar, 8Emanuelli M. Carnevali F. Lorenzi M. Raffaelli N. Amici A. Ruggieri S. Magni G. FEBS Lett. 1999; 455: 13-17Crossref PubMed Scopus (51) Google Scholar, 9Emanuelli M. Natalini P. Raffaelli N. Ruggieri S. Vita A. Magni G. Arch. Biochem. Biophys. 1992; 298: 29-34Crossref PubMed Scopus (41) Google Scholar, 10Emanuelli M. Carnevali F. Saccucci F. Pierella F. Amici A. Raffaelli N. Magni G. J. Biol. Chem. 2001; 276: 406-412Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 11Raffaelli N. Pisani F.M. Lorenzi T. Emanuelli M. Amici A. Ruggieri S. Magni G. J. Bacteriol. 1997; 179: 7718-7723Crossref PubMed Google Scholar). Along with sequence homology, the specificity of these enzymes for NMNversus NaMN provides a useful method for classifying new genes within this family. Although there is sequence conservation between the eubacterialnadD genes (Fig. 2), sequence alignment of nadD NaMN ATs to the eukaryotic enzymes or archeal enzymes is difficult outside of the region surrounding the (H/T)XGH nucleotidyl transferase consensus sequence. Adenylyltransferases encoded by the nadD gene prefer the nicotinic acid containing NaMN over NMN as a substrate by a factor that ranges from 6:1 to 2000:1 (4Magni G. Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 135-182PubMed Google Scholar, 12Mehl R.A. Kinsland C. Begley T.P. J. Bacteriol. 2000; 182: 4372-4374Crossref PubMed Scopus (41) Google Scholar, 13Begley T.P. Kinsland C. Mehl R.A. Osterman A. Dorrestein P. Vitam. Horm. 2001; 61: 103-119Crossref PubMed Google Scholar). Eubacteria also contain enzymes that demonstrate higher specificity for the nicotinamide-containing NMN. This group includes the products of the nadR gene, which in addition to its regulatory role in NAD biosynthesis also displays NMN AT activity (14Raffaelli N. Lorenzi T. Mariani P.L. Emanuelli M. Amici A. Ruggieri S. Magni G. J. Bacteriol. 1999; 181: 5509-5511Crossref PubMed Google Scholar). The eukaryotic and archeal NMN AT (EC 2.7.7.1), such as those from human (15Schweiger M. Hennig K. Lerner F. Niere M. Hirsch-Kauffmann M. Specht T. Weise C. Oei S.L. Ziegler M. FEBS Lett. 2001; 492: 95-100Crossref PubMed Scopus (105) Google Scholar),Methanococcus jannaschii (16D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Structure Fold Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (66) Google Scholar), and Methanobacterium thermoautotrophicum (17Saridakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276: 7225-7232Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), either demonstrate higher specificity for NMN as a substrate as compared with NaMN, or show little preference for either substrate (4Magni G. Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 135-182PubMed Google Scholar). Primary sequence studies indicate that NaMN AT belongs to the nucleotidyl-transferase α/β phosphodiesterases superfamily of enzymes that contain the (H/T)XGH signature motif. Members of this family share the same basic catalytic mechanism, involving direct nucleophilic attack upon an α-phosphate followed by the release of pyrophosphate, whereas the enzyme provides stabilization of the transition state prior to the formation of a new phosphodiester bond. The recent structure determination of NMN ATs from M. jannaschii and M. thermoautotrophicum has allowed this sequence and functional homology to be extended to the structural conservation of residues involved in substrate binding and catalysis (16D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Structure Fold Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (66) Google Scholar, 17Saridakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276: 7225-7232Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Genes that have been identified to be essential for bacterial cell survival are currently being evaluated for their potential as targets for anti-microbial chemotherapy. Understanding the biochemical, physical, and structural properties of these essential enzymes and placing them in a larger biological context are the first steps in exploring this potential. To this end, we report here the identification of an unassigned reading frame in Bacillus subtilis (yqeJ) as a NaMN AT. We have expressed the recombinant enzyme in Escherichia coli and show that it prefers NaMN as a substrate to NMN, allowing us to assign it as thenadD gene of B. subtilis. It differs from the NMN ATs from M. jannaschii and M. thermoautotrophicumboth in its substrate specificity and oligomeric state. It is dimeric as opposed to hexameric (16D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Structure Fold Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (66) Google Scholar, 17Saridakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276: 7225-7232Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We have determined the three-dimensional structure of NaMN AT from B. subtilis to 2.2Å and 3.2Å with the NaAD bound. This has allowed us to identify key residues in substrate binding and catalysis. These structures will provide invaluable information in the ongoing development of anti-microbial agents targeting NAD biosynthesis. The B. subtilis yqeJ gene was PCR-cloned into a modified version of pET16b to yield pML208. This E. coli expression vector has the yqeJ coding sequence downstream of the T7 RNA polymerase promoter. The expressed protein contains the peptide MGHHHHHHHHHHSSGHIEGRHMPGGS fused to Lys2 of the native sequence. This provides a purification tag and contains the cleavage site for Factor Xa between Arg20 and His21 of the peptide, resulting in the cleaved protein having six extra amino acids on its N terminus. To produce selenomethionine-labeledyqeJ, the protein was expressed in BL21(DE3) E. coli at 25 °C. The cultures were grown in shake flasks in LeMaster's medium and induced at log phase with 0.5 mmisopropyl-1-thio-β-d-galactopyranoside. The cultures were harvested 4 h post-induction. Unlabeled yqeJ was also expressed in BL21DE3 E. coli, but at 37 °C. High density expression was carried out in a Biostat C-10 bioreactor (B. Braun Biotech). The culture was induced with 1.0 mmisopropyl-1-thio-β-d-galactopyranoside (final) at 4.6A600. The cells were harvested 4 h post-induction at 9.0 A600. The purification, unless otherwise stated, was performed at 4 °C. Bacteria were resuspended in buffer (50 mm Hepes, pH 7.5, 500 mm NaCl) and lysed by passage through a Microfluidics microfluidizer. The lysate was collected and centrifuged at 20,000 × g for 30 min. The supernatant, containing 40% of the expressed yqeJ NaMN AT (the remainder being insoluble), was applied to a Poros PI column (Applied Biosystems) that was coupled to a nickel-nitrilotriacetic acid column (Qiagen). The nickel-nitrilotriacetic acid column was washed with 50 mmimidazole, and the protein was eluted with a 50–800 mmimidazole gradient. 10 mm EDTA was added to the fraction containing yqeJ NaMN AT, for 6 h, followed by dialysis against 50 mm Tris, pH 8.0, 50 mm NaCl, 2 mm CaCl2. The His tag was removed fromyqeJ NaMN AT by a 6-h digestion with Factor Xa (New England Biolabs) at room temperature. The reaction was applied to a Poros HQ 50 column, and the bound protein was eluted with a 0–1 m NaCl gradient. The peak fraction containing yqeJ NaMN AT was diluted and applied to a Poros S column. The flow through, containingyqeJ NaMN AT, was applied to TSK gel G3000 SW column (TosoHaas), equilibrated with 50 mm Hepes, pH 7.5, 50 mm NaCl. Protein purity was >95%. Discontinuous HPLC assay. The discontinuous HPLC assay is based upon the assays published by Mehlet al. (12Mehl R.A. Kinsland C. Begley T.P. J. Bacteriol. 2000; 182: 4372-4374Crossref PubMed Scopus (41) Google Scholar) and Balducci et al. (18Balducci E. Emanuelli M. Raffaelli N. Ruggieri S. Amici A. Magni G. Orsomando G. Polzonetti V. Natalini P. Anal. Biochem. 1995; 228: 64-68Crossref PubMed Scopus (67) Google Scholar). The reaction conditions were 20 mm Hepes, pH 7.4, 10 mm MgCl2, and 0.36 or 0.18 mg/mlyqeJ protein incubated at 37 °C. For the forward reaction, the incubations contained 1 mm ATP and 1 mm NaMN or NMN. For the reverse reaction, the incubations contained 1 mm sodium pyrophosphate and 0.5 mmNaAD or NAD. The reactants and products were separated by chromatography on a 3.9 × 150-mm C18 column (Novapack 5 μm; Waters Inc). Buffer A was 100 mm potassium phosphate, pH 7.5. Buffer B was 100 mm potassium phosphate, pH 7.5, in 20% MeOH. The elution conditions were: 0–3 min in 100% buffer A, 3.0–3.1 min to 100% buffer B, and 3.1–7 min in 100% buffer B. The absorbance of reactants and products was detected at 254 nm. Under these conditions NaMN eluted at 1.16 min, NMN eluted at 1.29 min, ATP eluted at 2.12 min, NaAD eluted at 5.37 min, and NAD eluted at 5.47 min. A continuous assay to monitor the reaction in the forward direction was based upon the EnzChek pyrophosphate assay from Molecular Probes (Eugene, OR). In this assay inorganic pyrophosphate produced in the forward reaction of yqeJ is cleaved by inorganic pyrophosphatase to phosphate, which is used by the second coupling enzyme, purine nucleoside phosphorylase, to convert the chromogenic substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside to ribose-1-phosphate and 2-amino-6-mercapto-7-methylpurine (E360 = 11,000 m−1cm−1). The reaction conditions were 20 mmHepes, pH 7.5, 10 mm MgCl2, 0.2 mm2-amino-6-mercapto-7-methylpurine ribonucleoside, 1 unit of purine nucleoside phosphorylase, 0.01 unit of inorganic pyrophosphatase, and 0.03 μg/ml yqeJ. The reaction volume was 125 μl and was carried out in a 96-well plate at room temperature using a Spectomax 384 Plus plate reader recording continuously at 360 nm (Molecular Devices, Sunnyvale, CA). For the Km determinations, NaMN was varied from 25 to 500 μm with ATP held at 2 mm, and ATP was varied from 50 to 1000 μmwith NaMN held constant at 1 mm. All kinetic constants were determined from nonlinear fits of the experimental data using the enzyme kinetic module of Sigmaplot 7.0 (SPSS, Inc., San Rafael, CA). The reaction conditions were the same for the Kmdeterminations for NMN except the MgCl2 concentration was increased to 50 mm, and the yqeJ concentration was increased to 75 μg/ml because of the lower activity against this substrate. Substrate inhibition was seen in assays using NMN as a substrate, with the double reciprocal plots curving sharply upwards above 5 mm. The data were fit to a model of substrate inhibition using the equation: v =Vmax/(1 + Km/S +S/Ki). The Kidetermined for NMN was 17 ± 3 mm. NMN was varied from 0.5 to 10 mm with ATP held constant at 0.6 mm. We also determined the kinetic constants of yqeJ using NMN and ATP substrates with a second coupled assay system, one coupling NAD production to its reduction by alcohol dehyrogenase. This assay was described by Balducci et al. (5Balducci E. Orsomando G. Polzonetti V. Vita A. Emanuelli M. Raffaelli N. Ruggieri S. Magni G. Natalini P. Biochem. J. 1995; 310: 395-400Crossref PubMed Scopus (27) Google Scholar) and has been used to characterize other NMN ATase. Using this assay we obtained values ofKm and Vmax for NMN very similar to those determined with the purine nucleoside phosphorylase-coupled assay described above. The alcohol dehydrogenase-coupled assay was not suitable to assay NaMN as a substrate, presumably because NaAD is not a good substrate for alcohol dehydrogenase. The back reaction for yqeJ NaMN AT was monitored using the coupled enzyme assay of hexokinase and glucose-6-phosphate dehydrogenase (from yeast). yqeJ NaMN AT converts pyrophosphate and NaAD (or NAD) to NaMN (or NMN) and ATP. The ATP is then used by hexokinase to phosphorylate glucose to give glucose-6-phosphate and ADP. Glucose-6-phosphate is oxidized to 6-phospho-glucono-δ-lactone by glucose-6-phosphate dehydrogenase, and NADP is reduced to NADPH. The assay is followed by the absorbance of NADPH at 340 nm. We used glucose-6-phosphate dehydrogenase from bakers' yeast because this enzyme prefers NADP to NAD as a co-substrate. The assay conditions were: 20 mm Hepes, pH 7.5, 50 mm MgCl2, 1 mm NaPPi, 10 mm KCl, 5 units of hexokinase, 5 units of glucose-6-phosphate dehydrogenase, 1 mm glucose, and 0.5 mm NADP+. yqeJ concentration is 0.0015 mg/ml for NaAD determination and 0.0075 mg/ml for NAD determination. ForKm determinations, NaAD was varied from 5 to 100 μm holding PPi at 1 mm, NAD was varied from 1 to 10 mm holding PPi at 1 mm, and PPi was varied from 0.1 to 2 mm holding NaAD at 500 μm. At a high concentration of NaPPi a precipitate was observed. This limited the concentrations of PPi that could be used in the assays. Crystals were grown by hanging drop vapor diffusion at 18 °C in drops containing 1.5 μl of protein stock solution (14 mg/ml protein, 50 mm Hepes, pH 7.5, 50 mm NaCl) mixed with 1.5 μl of well solution (8% PEG 3350, 100 mm MgCl2) and equilibrated against 1 ml of well solution. Block shaped crystals grew in 3 weeks, measuring ∼50 μm across. NaAD-NaMN AT complex co-crystals were grown at 18 °C in drops containing 1.0 μl of protein stock solution (14 mg/ml protein, 2 mm NaAD, 50 mm Hepes, pH 7.5, 50 mm NaCl) mixed with 1.0 μl of well solution (20% PEG 3350, 100 mm magnesium acetate) and 0.3 μl of xylitol (30% w/v). Plate-like crystals grew in 1–3 weeks to ∼200 × 50 × 20 μm. Crystals of the apo-form belong to the space group P21 with unit cell parametersa = 43.98 Å, b = 126.10 Å,c = 70.58 Å, and β = 92.73° and contain four molecules of NaMN AT in the asymmetric unit, implying a solvent content of 58.5%. To harvest crystals, an equal volume of a solution of 35% PEG 3350, 100 mm MgCl2 was added to drops, and after equilibration for several minutes the crystals were swiped through another drop of this solution and cooled rapidly in liquid nitrogen. The data collection statistics are shown in Table II. Multiple wavelength anomalous dispersion (MAD) data were recorded at the 5.0.2 beam line of the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory using a Quantum-4 detector. The data were collected at two energies chosen based on the measured absorption at the selenium K edge: 12661 eV (λ = 0.9792 Å) and 12959 eV (λ = 0.9567 Å) corresponding to maximum f" and a remote energy, respectively. Intensities were integrated and scaled using the programs Denzo and Scalepack (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38445) Google Scholar).Table IIStatistics for data collection, phase determination, and refinementData collectionPeakRemoteEmpty Wavelength (Å)0.97920.9567 Resolution range (Å)20–2.220–2.0 Rmerge(%)2-aRmerge = ‖Ih − 〈Ih〉‖/Ih, where 〈Ih〉 is the average intensity over symmetry equivalents. The numbers in parentheses reflect the statistics for the last shell.10.3 (45.3)7.9 (47.0) Completeness (%)100.0 (100.0)99.9 (99.8) Total reflections286583196259 Unique reflections3871051680 I/ς(I)22.7 (3.3)16.4 (2.4)NaAD-bound Wavelength (Å)0.97910.9639 Resolution range (Å)15.0–3.215.0–3.4 Rmerge(%)2-aRmerge = ‖Ih − 〈Ih〉‖/Ih, where 〈Ih〉 is the average intensity over symmetry equivalents. The numbers in parentheses reflect the statistics for the last shell.14.7 (28.9)11.1 (27.6) Completeness (%)96.5 (84.0)96.8 (79.0) Total reflections8049999889 Unique reflections2459920850 I/ς(I)16.0 (5.6)12.3 (4.8)Model refinementNaMN ATNAAD-NaMN ATMaximum resolution (Å)2.13.2Rwork(%)2-bRwork = ∥Fobs‖ − ‖Fcalc∥/‖Fobs‖.22.2125.64Rfree(%)2-cRfree is equivalent toRwork but calculated for a randomly chosen 5% of reflections omitted from the refinement process.25.4429.75RMS deviations from ideal geometry For bonds (Å)0.0090.012 For angles (°)1.3471.913Nonhydrogen protein atoms78489051Water molecules13902-a Rmerge = ‖Ih − 〈Ih〉‖/Ih, where 〈Ih〉 is the average intensity over symmetry equivalents. The numbers in parentheses reflect the statistics for the last shell.2-b Rwork = ∥Fobs‖ − ‖Fcalc∥/‖Fobs‖.2-c Rfree is equivalent toRwork but calculated for a randomly chosen 5% of reflections omitted from the refinement process. Open table in a new tab Crystals of the NaAD-bound form belong to the space group P212121 with unit cell parametersa = 78.39 Å, b = 108.90 Å,c = 178.09 Å, and α = β = γ = 90.00° and contain six molecules of yqeJ in the asymmetric unit, implying a solvent content of 55.6%. To harvest crystals, ethylene glycol was added to the drops to 20%, and after mixing, the crystals were cooled rapidly in liquid nitrogen. The data collection statistics are shown in Table II. MAD data were recorded at the 5.0.2 beam line of the Advanced Light Source at Lawrence Berkeley National Laboratory using a Quantum-4 detector. The data were collected at two consecutive energies, based on the measured absorption at the selenium K edge: 12662 eV (λ = 0.9791Å) and 12863 eV (λ = 0.9639Å) and corresponding to maximum f" and a remote energy. Intensities were integrated and scaled using the programs Denzo and Scalepack (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38445) Google Scholar). The apo structure was determined by the MAD method. Initially 22 selenium sites were found with the program SOLVE (20Terwilliger T.C. Berendzen J. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar), and phasing with these sites in the program CNS (21Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16947) Google Scholar) revealed two additional sites by means of a difference Fourier map. Phases were calculated from these 24 sites in the programs CNS and SHARP (22De La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 4772-4794Google Scholar) and improved by solvent modification with the program Solomon (22De La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 4772-4794Google Scholar). ARP/WARP (23Perrakis A. Morris R.J. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar) was used to partially trace the α-carbon backbone. The complete model, with the exception of residues 42–52 in all molecules and residues 118–126 in molecules A and C, was built into the experimental map. After several iterative cycles of refinement using CNS and model improvement, water molecules were placed automatically in CNS. A simulated annealing composite omit map was calculated to check the final model. FinalRwork and Rfree values of 22.21 and 25.44% were obtained. The NaAD-NaMN AT complex structure was determined using a combination of MAD phases and molecular replacement using the apo enzyme structure. Initially 21 selenium sites were found using the program ShakeNBake (24Weeks C.M. Miller R. J. Appl. Crystallogr. 1999; 32: 120-124Crossref Scopus (384) Google Scholar), and phasing with these sites in the program MLPhare (25CCP4 Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19730) Google Scholar) revealed 13 additional sites by means of a difference Fourier map. Phases were calculated from these 34 sites and improved with solvent flattening and 4-fold averaging with the program DM (25CCP4 Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19730) Google Scholar). This experimental map was used to build four of the six molecules in the asymmetric unit, employing strict non-crystallographic symmetry restraints. At this point, the refined structure of the apo-enzyme became available. The complex model was edited such that fragments of the high resolution apo model were substituted wherever the apo model was in good agreement with electron density in the experimental map. MAD phases were then combined with model phases to generate improved maps, and two more molecules were identified. Density for the first four molecules found is significantly stronger than for the final two; however, density for the bound NaAD is good for all 6 molecules. The final Rvalues after releasing NCS restraints and rebuilding areRfree = 0.2798 and Rwork= 0.2787. 90 μg of B. subtilis NaMN AT was run on an Amersham Biosciences, Inc. Superose 12 H/R 10/30 column in Tris-buffered saline, pH 7.5, at a flow rate of 0.5 ml/min. The retention time of B. subtilis NaMN AT was 27.3 min. To determine the apparent molecular weight of B. subtilis NaMN AT, the following standards were also run on the column under the same conditions: thyroglobin, 670,000; Gamma globulin, 158,000; ovalbumin, 44,000; myoglobin, 17,000; and vitamin B-12, 1,350 (Bio-Rad). Protein was pre-equilibrated by dialysis at 4 °C in 20 mm Hepes and 10 mmMgCl2, pH 7.2. The partial specific volume of B. subtilis NaMN AT was calculated based on the amino acid composition, and the density of the solvent was calculated from the chemical composition of the buffer using the computer program SEDNTERP and adjusted for temperature. Sedimentation velocity experiments were performed on a Beckman XLI/XLA Analytical Ultracentrifuge operating at a rotor speed of 30,000 rpm using 400-μl samples loaded into two-channel carbon-Epon centerpieces in an An-60 Ti titanium rotor pre-equilibrated to temperature at least 1 h prior to each experiment. The sedimentation coefficients and molecular weights were obtained by fitting the data to the program SVEDBERG (26Philo J.S. Schuster T.M. Lane T.M. Modern Analytical Ultracentrifugation. Birkhauser, Boston, MA1994: 156-170Crossref Google Scholar). Sedimentation equilibrium experiments were performed at 4 and 20 °C using a rotor speed of 18,000 rpm. The samples (400 μl) were loaded into two-channel cells at three different protein concentrations. The scans were recorded at 4 and 20 °C, and the signal was detected using absorbance optics (280 nm) and interference optics. Equilibrium was judged to be achieved when no deviations in a plot of the difference between successive scans taken 3 h apart were observed, usually within 24 h. However, temperature and ligand did have a minor effect upon the apparent molecular weight as determined by analytical ultracentrifugation. The variation between the velocity and equilibrium values at the different protein concentrations can be attributed to hydrodynamic effects. The molecular weight of the protein in the presence and absence of ligand was obtained from sedimentation equilibrium experiments using the following equation. Cr=C0expM(1−v¯ρ)ω2r2−r02/2RT+baseEquation 1 Where Cr is the absorbance at radiusr; C0 is the absorbance at reference radius r0; M is the molar mass of the macromolecule; v̄ is the partial specific volume of the macromolecule (ml/g); ρ is the density of the solvent; ω is the angular velocity of the rotor; R is the gas constant;T is the temperature; and base is the base-line offset. The molecular weight of yqeJ NaMN AT was obtained from sedimentation velocity experiments using Equation 2.M=(sRT)/(1−v¯ρ)DEquation 2 Where s is the sedimentation coefficient andD is the diffusion coefficient obtained by fitting the data to the program SVEDBERG (26Philo J.S. Schuster T.M. Lane T.M. Modern Analytical Ultracentrifugation. Birkhauser, Boston, MA1994: 156-170Crossref Google Scholar). The Streptococcus pneumonia genome was sequenced and searched for essential genes that may be suitable targets for the development of anti-microbial agents. Those genes identified were then tested in B. subtilis and E. coli. An unassigned open reading frame, yqeJ, that was essential in all three organisms was identified in B. subtilis. Comparisons of its amino acid sequence with those in GenBankTM using the program Blast (27Altschol S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69694) Google Scholar) revealed that it has homology to a number of putative adenylyltransferases including the recently assigned nadD gene of E. coli (12Mehl R.A. Kinsland C. Begley T.P. J. Bacteriol. 2000; 182: 4372-4374Crossref PubMed Scopus (41) Google Scholar).yqeJ contains the signature nucleotidyl transferase consensus sequence (H/T)XGH. As can be seen in Fig. 2,B. subtilis yqeJ is closely related to E. coli nadD and other putative eubacterial NaMN ATs. TheB. subtilis enzyme is more distantly related to E. coli nadR and other eukaryotic and archeal NMN ATs. Alignment of the B. subtilis NaMN AT to these species was difficult because of little homology outside of the region around the (H/T)XGH consensus sequence. This later group of enzymes includes the NMN AT from M. jannaschii and M. thermoautotrophicum for which three-dimensional structures have recently been determined (16D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Structure Fold Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (66) Google Scholar, 17Saridakis V. Christendat D. Kimber M.S. Dharamsi A. Edwards A.M. Pai E.F. J. Biol. Chem. 2001; 276