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• W2080303413 abstract "We have determined the crystal structure of nicotinate phosphoribosyltransferase from Themoplasma acidophilum (TaNAPRTase). The TaNAPRTase has three domains, an N-terminal domain, a central functional domain, and a unique C-terminal domain. The crystal structure revealed that the functional domain has a type II phosphoribosyltransferase fold that may be a common architecture for both nicotinic acid and quinolinic acid (QA) phosphoribosyltransferases (PRTase) despite low sequence similarity between them. Unlike QAPRTase, TaNAPRTase has a unique extra C-terminal domain containing a zinc knuckle-like motif containing 4 cysteines. The TaNAPRTase forms a trimer of dimers in the crystal. The active site pocket is formed at dimer interfaces. The complex structures with phosphoribosylpyrophosphate (PRPP) and nicotinate mononucleotide (NAMN) showed, surprisingly, that functional residues lining on the active site of TaNAPRTase are quite different from those of QAPRTase, although their substrates are quite similar to each other. The phosphate moiety of PRPP and NAMN is anchored to the phosphate-binding loops formed by backbone amides, as found in many α/β barrel enzymes. The pyrophosphate moiety of PRPP is located at the entrance of the active site pocket, whereas the nicotinate moiety of NAMN is located deep inside. Interestingly, the nicotinate moiety of NAMN is intercalated between highly conserved aromatic residues Tyr21 and Phe138. Careful structural analyses combined with other NAPRTase sequence subfamilies reveal that TaNAPRTase represents a unique sequence subfamily of NAPRTase. The structures of TaNAPRTase also provide valuable insight for other sequence subfamilies such as pre-B cell colony-enhancing factor, known to have nicotinamide phosphoribosyltransferase activity. We have determined the crystal structure of nicotinate phosphoribosyltransferase from Themoplasma acidophilum (TaNAPRTase). The TaNAPRTase has three domains, an N-terminal domain, a central functional domain, and a unique C-terminal domain. The crystal structure revealed that the functional domain has a type II phosphoribosyltransferase fold that may be a common architecture for both nicotinic acid and quinolinic acid (QA) phosphoribosyltransferases (PRTase) despite low sequence similarity between them. Unlike QAPRTase, TaNAPRTase has a unique extra C-terminal domain containing a zinc knuckle-like motif containing 4 cysteines. The TaNAPRTase forms a trimer of dimers in the crystal. The active site pocket is formed at dimer interfaces. The complex structures with phosphoribosylpyrophosphate (PRPP) and nicotinate mononucleotide (NAMN) showed, surprisingly, that functional residues lining on the active site of TaNAPRTase are quite different from those of QAPRTase, although their substrates are quite similar to each other. The phosphate moiety of PRPP and NAMN is anchored to the phosphate-binding loops formed by backbone amides, as found in many α/β barrel enzymes. The pyrophosphate moiety of PRPP is located at the entrance of the active site pocket, whereas the nicotinate moiety of NAMN is located deep inside. Interestingly, the nicotinate moiety of NAMN is intercalated between highly conserved aromatic residues Tyr21 and Phe138. Careful structural analyses combined with other NAPRTase sequence subfamilies reveal that TaNAPRTase represents a unique sequence subfamily of NAPRTase. The structures of TaNAPRTase also provide valuable insight for other sequence subfamilies such as pre-B cell colony-enhancing factor, known to have nicotinamide phosphoribosyltransferase activity. NAD is an essential cofactor for both energy metabolism and signal transduction similar to dual functional nucleotides, ATP and GTP. The role of NAD acting on redox equilibrium in metabolism has been well known in processes such as DNA repair and calcium-dependent signaling pathways (1Lin S.J. Guarente L. Curr. Opin. Cell Biol. 2003; 15: 241-246Crossref PubMed Scopus (403) Google Scholar). NAD is synthesized via two major pathways in both prokaryotic and eukaryotic organisms. In one pathway, NAD is synthesized from tryptophan, a de novo pathway (2Kucharczyk R. Zagulski M. Rytka J. Herbert C.J. FEBS Lett. 1998; 424: 127-130Crossref PubMed Scopus (48) Google Scholar), and in the other, NAD is generated by recycling degraded NAD products such as nicotinamide, referred to as the salvage pathway (3Tritz G.J. Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1987: 557-563Google Scholar). The three precursors, quinolinate (QA), 1The abbreviations used are: QA, quinolinic acid; PRTase, phosphoribosyltransferase; MtQAPRTase, QAPRTase from M. tuberculosis; StQAPRTase, S. typhimurium; PRPP, phosphoribosylpyrophosphate; NA, nicotinate; NAMN, nicotinate mononucleotide; NAD, nicotinamide adenine dinucleotide; NAm, nicotinamide; NAPRTase, NA phosphoribosyltransferase; TaNAPRTase, NAPRTase from T. acidophilum; St- NAPRTase, NAPRTase from S. typhimurium; EcNAPRTase, NAPRTase from E. coli; NAmPRTase, nicotinamide phosphoribosyltransferase; PBEF, pre-B-cell colony-enhancing factor; PDB, Protein Data Bank. nicotinate (NA), and nicotinamide (NAm), can be transferred onto phosphoribosyl pyrophosphate (PRPP) by the respective phosphoribosyl transferase (Fig. 1). The resulting mononucleotides, nicotinamide mononucleotide or nicotinic acid mononucleotide (NAMN), are converted into the corresponding dinucleotides, NAD or nicotinic acid adenine dinucleotide, by nicotinamide mononucleotide adenylyltransferase (4Berger F. Ramirez-Hernandez M.H. Ziegler M. Trends Biochem. Sci. 2004; 29: 111-118Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). Finally, nicotinic acid adenine dinucleotide is amidated to NAD by NAD synthase. Surprisingly, increasing the activity of NAD biosynthetic enzymes has recently been demonstrated to extend life span (5Denu J.M. Trends Biochem. Sci. 2003; 28: 41-48Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 6Hekimi S. Guarente L. Science. 2003; 299: 1351-1354Crossref PubMed Scopus (378) Google Scholar). Overexpression of enzymes of the NAD biosynthetic pathway such as nicotinate phosphoribosyltransferase (NAPRTase), nicotinate mononucleotide adenylyltransferase, or nicotinamidase in yeast results in higher activity of the NAD-dependent histone deacetylase Sir2p and concomitant life span extension (7Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Sinclair D.A. Nature. 2003; 423: 181-185Crossref PubMed Scopus (612) Google Scholar, 8Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1488) Google Scholar, 9Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Cohen H. Lin S.S. Manchester J.K. Gordon J.I. Sinclair D.A. J. Biol. Chem. 2002; 277: 18881-18890Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). NAPRTase is a facultative ATPase that couples ATP hydrolysis and nucleotide formation in the salvage pathway (3Tritz G.J. Neidhardt F.C. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1987: 557-563Google Scholar). The NAPRTase from Salmonella typhimurium (StNAPRTase) catalyzes NAMN formation without ATP but accelerates the reaction 10-30-fold in its presence (10Gross J.W. Rajavel M. Grubmeyer C. Biochemistry. 1998; 37: 4189-4199Crossref PubMed Google Scholar). A low affinity form of StNAPRTase is phosphorylated by ATP followed by conversion to a high affinity form binding substrates and producing NAMN. The covalently phosphorylated site, His219, is converted to a labile 1-phosphohistidine by ATP (11Rajavel M. Lalo D. Gross J.W. Grubmeyer C. Biochemistry. 1998; 37: 4181-4188Crossref PubMed Google Scholar). To elucidate the molecular function of NAPRTase, we have determined the crystal structures of native and substrate-bound forms of an NAPRTase homologue from Thermoplasma acidophilum (TaNAPRTase, gi: 16082162). In the Pfam data base (12Bateman A. Birney E. Durbin R. Eddy S.R. Howe K.L. Sonnhammer E.L. Nucleic Acids Res. 2000; 30: 276-280Crossref Scopus (2021) Google Scholar), TaNAPRTase belongs to pfam04095 together with StNAPRTase and EcNAPRTase (NAPRTase from Escherichia coli). The TaNAPRTase also shows strong sequence homology to pre-B-cell colony-enhancing factor (PBEF), one of the members of Pfam04095 (Fig. 2A). The murine homologue of the previously identified human PBEF gene coding for a putative cytokine has been identified as a nicotinamide phosphoribosyltransferase (NAmPRTase), indicating that NAD biosynthesis may play an important role in lymphocyte activation (13Rongvaux A. Shea R.J. Mulks M.H. Gigot D. Urbain J. Leo O. Andris F. Eur. J. Immunol. 2002; 32: 3225-3234Crossref PubMed Scopus (481) Google Scholar). The structural and functional relationship of NAPRTase sequence subfamilies is also discussed based on the crystal structures together with sequence comparisons.Fig. 2Sequence comparisons. A, comparison of sequences and secondary structures between NAPRTase and QAPRTases. Abbreviations are as follows: human (Hs), Rattus norvegicus (Rn), Thermotoga maritima (Tm). For others, refer to the abbreviations used. The secondary structure prediction results of StNAPRTase, EcNAPRTase, HsPBEF, and RnPBEF were obtained from PSIPRED (bioinf.cs.ucl.ac.uk/psipred). The crystal structures of MtQAPRTase (PDB accession code: 1qpn) and TmQAPRTase (1o4u) were used to derive their secondary structure elements. Blue characters represent α-helix, red for β-strand, and green characters represent 310-helix. Only the type II phosphoribosyltransferase fold is represented with symbols of α-helix and β-strand based on the MtQAPRTase structure. B, Sequence comparison between TaNAPRTase and its homologues. Abbreviations are as follows: T. acidophilum (Ta), Thermoplasma volcanium (Tv), Archaeoglobus fulgidus (Af), Halobacterium sp. NRC-1 (Hn), P. horikoshii (Ph), Pyrococcus furiosus DSM 3638 (Pf), Aeropyrum pernix (Ap), Aquifex aeolicus (Aa). Domains are represented as follows: red for the N-terminal domain, green for the central domain, and yellow for the C-terminal zinc knuckle-like domain. Secondary structure elements are represented above the sequence alignment. Blue represents the α-helix, red diamonds represent the β-strand, and green represents the 310-helix. The - - - represents a gap, the * represents identical residues, the: represents highly conserved residues, and the · represents less highly conserved residues. The percentage of sequence identities are also shown. DomI represents sequence identity of domain I of each species against TaNAPRTase, DomII represents domain II, DomIII represents domain III, and ALL represents the entire protein, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cloning of TaNAPRTase—The sequence encoding TaNAPRTase was amplified by the PCR from T. acidophilum genomic DNA (American Type Culture Collection, Manassas, VA) using Deep Vent DNA polymerase (New England Biolabs, Beverly, MA). The resulting PCR product was purified and prepared for ligation-independent cloning (14Aslanidis C. de Jong P.J. Nucleic Acids Res. 1990; 20: 6069-6074Crossref Scopus (948) Google Scholar) by treatment with T4 DNA polymerase in the presence of 1 mm dTTP for 30 min at 37 °C. The prepared DNA was then mixed with the vector pB4 for 5 min at room temperature and transformed into DH5α. This pB4 vector was designed in our laboratory to express the target protein together with an N-terminal His6 tag-maltose-binding protein fusion containing a tobacco etch virus protease cleavage site. Clones were screened by plasmid DNA analysis and transformed into BL21(DE3)/pSJS1244 for protein expression (15Kim R. Sandler S.J. Goldman S. Yokota H. Clark A.J. Kim S-H. Biotechnol. Lett. 1998; 20: 207-210Crossref Scopus (138) Google Scholar). Protein Expression, Purification, and Crystallization—A selenomethionine derivative of the protein was expressed in a methionine auxotroph, E. coli strain B834(DE3)/pSJS1244 (15Kim R. Sandler S.J. Goldman S. Yokota H. Clark A.J. Kim S-H. Biotechnol. Lett. 1998; 20: 207-210Crossref Scopus (138) Google Scholar), grown in PASM medium 2W. Studier, personal communication. supplied with selenomethionine (16Leahy D.J. Hendrickson W.A. Aukhil I. Erickson H.P. Science. 1992; 258: 987-991Crossref PubMed Scopus (447) Google Scholar). Cells were disrupted by microfluidization (Microfluidics, Newton, MA) in 50 mm HEPES, pH 7, 500 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml DNase, 0.1 μg/ml antipain, 1 μg/ml chymostatin, 0.5 μg/ml leupeptin, and 0.7 μg/ml pepstatin A, and cell debris was pelleted by centrifugation at 10,000 rpm for 20 min in a Sorvall centrifuge. The supernatant was then spun in a Beckman ultracentrifuge Ti45 rotor at 35,000 rpm for 30 min at 4 °C. The fusion protein was affinity-purified using two 5-ml HiTrap chelating HP columns (Amersham Biosciences). The fusion protein was bound to the column in 5% glycerol, 50 mm HEPES, pH 7.0, 500 mm NaCl and was eluted with a gradient of 4-300 mm imidazole in 10 column volumes. Fractions were pooled and dialyzed overnight at room temperature against 50 mm HEPES, pH 7.0, 0.1 m NaCl, 5 mm β-mercaptoethanol, and 10 mm imidazole in the presence of tobacco etch virus protease. After centrifugation, the supernatant was applied onto a 5-ml HiTrap metal chelating (Ni2+) column. The cleaved recombinant protein was found in the flow-through. Further purification was performed with size-exclusion chromatography in 20 mm Tris-HCl, pH 7.5, and 300 mm NaCl. SDS-PAGE showed one band ∼ 43 kDa, corresponding to the molecular mass of TaNAPRTase. The protein was concentrated to 8.1 mg/ml for crystallization. Dynamic light scattering (DynaPro 99, Wyatt Technology Corp., Santa Barbara, CA) showed a polydisperse peak, indicating the presence of an oligomeric form of TaNAPRTase. Screening for crystallization conditions was performed using the sparse matrix method (17Jancarik J. Kim S-H. J. Appl. Crystallogr. 1991; 24: 409-411Crossref Scopus (2076) Google Scholar) with several screens from Hampton Research (Hampton Research, Laguna Niquel, CA) and the Wizard Screen (deCODE genetics, Bainbridge Island, WA). The crystallization robot “Hydra Plus-One” (Matrix Technologies, Hudson, NH) was used to set the screens using the sitting drop vapor diffusion method at room temperature. In the optimized crystallization conditions, 1 μl of protein solution was mixed with 1 μl of the well solution containing 0.2 m ammonium acetate, 0.1 m Tris-HCl, pH 8.5, 45% 2-methyl-2,4-pentanediol, using the hanging drop vapor diffusion method. A thin rod crystal grew in a week to approximate dimensions of 0.08 × 0.02 × 0.02 mm3. The PRPP-bound complex was obtained by soaking a crystal with 10 mm PRPP for 12 h. When crystals are grown in 2.4 m sodium malonate at pH 7.0, the electron density map showed the presence of partially occupied NAMN. Interestingly, the addition of 10 mm nicotinate for 12 h clearly showed a fully occupied NAMN bound in the structure. Data Collection and Reduction—The crystal grown in the first crystallization solution was soaked in a drop of mother liquor containing 5 mm K2IrCl6 for 12 h and then transferred to mother liquor with 10% glycerol before being flash-frozen in liquid nitrogen and used for x-ray data collection. X-ray diffraction data to 2.8 Å were collected at a single wavelength (1.10490 Å, Table I) of iridium absorption peak at the Macromolecular Crystallography Facility beamline 5.0.2 at the Advanced Light Source at Lawrence Berkeley National Laboratory using an Area Detector System Co. (Poway, CA) Quantum 4 CCD detector placed 250 mm from the sample. The oscillation range per image was 1.0° with no overlap between two contiguous images. X-ray diffraction data were processed and scaled using DENZO and SCALEPACK from the HKL program suite (18Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The data sets for the complex crystals with PRPP and NAMN were collected to 2.75 and 2.65 Å, respectively. Data statistics are summarized in Table I.Table IStatistics of the peak wavelength single-wavelength anomalous dispersion phases data setData setIridium complexPRPP complexNAMN complexWavelength (Å)1.104901.282300.97950Resolution (Å)50.0−2.8050.0−2.7550.0−2.65RedundancyaNumbers in parenthesis refer to the highest resolution shell, which is 2.85−2.80, 2.80−2.75, and 2.70−2.65 Å, respectively., bRsym=∑hkl∑i|Ihkl,i−〈I〉hkl|/∑|Ihkl|.8.4 (8.7)12.5 (12.9)9.5 (9.2)Unique reflections16303 (796)16818 (830)19180 (941)Completeness (%)97.7 (97.9)96.2 (97.7)99.3 (99.9)I/σ16.0 (2.7)18.2 (3.4)19.0 (3.6)RsymaNumbers in parenthesis refer to the highest resolution shell, which is 2.85−2.80, 2.80−2.75, and 2.70−2.65 Å, respectively. (%)12.9 (84.7)16.0 (99.9)11.6 (82.4)a Numbers in parenthesis refer to the highest resolution shell, which is 2.85−2.80, 2.80−2.75, and 2.70−2.65 Å, respectively.b Rsym=∑hkl∑i|Ihkl,i−〈I〉hkl|/∑|Ihkl|. Open table in a new tab Structure Determination and Refinement—One major and two minor iridium positions were located using the program SOLVE (19Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar) with figure of merit 0.22 at 2.9 Å resolution. The initial single-wavelength anomalous dispersion phases were further improved by solvent flattening using the program RESOLVE (19Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The best interpretable map was found from 20.0 to 2.9 Å resolution data with figure of merit 0.55. There was one molecule in the asymmetric unit, and a model was built using the program O (20Jones A. Kleywegt G. Methods Enzymol. 1997; 277: 173-208Crossref PubMed Scopus (505) Google Scholar) with the aid of secondary structure prediction from PSIPRED (21Jones D.T. J. Mol. Biol. 1999; 292: 195-202Crossref PubMed Scopus (4500) Google Scholar). The preliminary model was then refined using the program CNS (22Brunger 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. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). The reflections in this data set between 20.0 and 2.8 Å were included throughout the refinement calculations. Ten percent of the data were randomly chosen for free R-factor cross validation. The refinement statistics are shown in Table II. Isotropic B-factors for individual atoms were initially fixed to 20 Å2 and were refined in the last stages. The 2Fo - Fc and Fo - Fc maps were used for manual rebuilding between refinement cycles and for the location of solvent molecules. Two complex structures containing PRPP and NAMN were solved at 2.75 and 2.65, respectively. Atomic coordinates of the three structures have been deposited in the Protein Data Bank (PDB) with the accession code of 1YTD, 1YTE, and 1YTK, respectively.Table IICrystal parameters and refinement statisticsData setIridium complexPRPP complexNAMN complexSpace groupP6322P6322P6322Cell dimensions (Å3)136.0 × 136.0 × 119.7136.1 × 136.1 × 120.2135.2 × 135.2 × 119.7β = 120β = 120β = 120Volume fraction of solvent65.4%65.6%65.0%Vn (Å3/dalton)3.693.713.65Total number of residues389389389Total non-H atoms305031093105Number of water molecules397664Average temperature factorsProtein45.9 Å240.2 Å247.4 Å2Solvent35.9 Å239.7 Å242.9 Å2OthersN/A93.8 Å261.7 Å2Resolution range of reflections used20.0−2.8 Å20.0−2.75 Å20.0−2.65 ÅAmplitude cutoff0.0 σ0.0 σ0.0 σR-factor19.8%19.6%20.1%Free R-factor24.6%25.4%25.3%Stereochemical idealityBond0.009 Å0.007 Å0.009 ÅAngle1.4°1.3°1.5°Improper0.86°0.89°1.03°Dihedral23.2°22.9°23.1° Open table in a new tab Quality of the Model and Overall Structure—The final model of the native crystal structure includes 389 out of 393 residues. The final models have been refined at 2.8 Å resolution to a crystallographic R-factor of 19.8% and free R-factor of 24.6% (Fig. 3). The averaged B-factors for main chain atoms and side chain atoms are 43.6 and 47.4 Å2, respectively. In the TaNAPRTase models, 3 C-terminal residues are undefined in the electron density map. Table II summarizes the refinement statistics as well as model quality parameters. All residues lie in the allowed region of the [phis] - φ plot produced with PROCHECK (23Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The Cα trace of the atomic model of TaNAPRTase is shown in Fig. 4A. The monomer has approximate dimensions of 80 Å × 55 Å × 25 Å. TaNAPRTase consists of three domains, an N-terminal α + β domain, a central α/β domain, and a C-terminal domain containing a zinc knuckle-like domain. The three dimers form a hexamer with a circular ring structure in the unit cell (Fig. 4, B-D). The thickness of the ring is about 50 Å, and the approximate diameters inside and outside of the ring are 35 and 90 Å, respectively. The presence of quaternary structures is supported by results from a dynamic light scattering experiment indicating the presence of several oligomeric states of TaNAPRTase in solution (data not shown). The surface area of TaNAPRTase buried by dimer formation is very large (per monomer ∼2,730 Å2). Interestingly, the major interaction at the dimeric interface is ionic rather than hydrophobic, as usually found in dimeric proteins. The interaction between dimers (∼1,950 Å2/dimer) that results in a hexameric state in the crystal structure is also ionic. Therefore, the oligomeric states can be achieved or destroyed depending on surrounding conditions influencing these ionic interactions such as pH, salt, or protein concentration. The result from a size-exclusion column in high salt conditions proved that TaNAPRTase could be present as a monomer (see “Materials and Methods”). A similar hexamer formation by three dimers dependent on high protein concentration or ionic strength has been reported based on the crystal structure of QAPRTase from Mycobacterium tuberculosis (MtQAPRTase) (24Sharma V. Grubmeyer C. Sacchettini J.C. Structure. 1998; 6: 1587-1599Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The major interaction at the dimeric interface of the MtQAPRTase is also ionic rather than hydrophobic. The final models of a NAMN-bound and PRPP-bound structures have been determined at 2.65 and 2.75 Å, respectively. The difference Fourier maps were used to build substrate models. In the PRPP-bound structure, the electron density of a pyrophosphate moiety was weak (Fig. 3A). This might be the result of the lack of magnesium ion known to bind with PRPP. In the NAMN-bound structure, in addition to NAMN-bound in the active site, one Tris buffer molecule was also found in the vicinity of Lys122 and Lys125 in the refined model. Table II summarizes the refinement statistics as well as model quality parameters. All residues lie in the allowed region of the [phis] - φ plot produced with PROCHECK (23Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). These substrate-bound crystal structures suggested that the functional unit of TaNAPRTase is at least a dimeric form as discussed below. Overall Structural Comparison of TaNAPRTase with QAPRTases—A DALI search (25Holm L. Sander C. Nucleic Acids Res. 1997; 25: 231-234Crossref PubMed Scopus (361) Google Scholar) shows that the highest similarity is found with MtQAPRTase (24Sharma V. Grubmeyer C. Sacchettini J.C. Structure. 1998; 6: 1587-1599Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) with Z-value of 18.9 (root mean square deviations of 2.9 Å for 254 pairs of aligned Cα atoms; 1qpo-A). Most of the N- and the central domains of TaNAPRTase are matched with the whole domain of MtQAPRTase (Fig. 5A), despite the low sequence similarity between NAPRTase and QAPRTase (Fig. 2A). The N-terminal domain of TaNAPRTase is composed of a β-sheet stacked with four α-helices having an α-β plait topology similar to that of MtQAPRTase. However, the following differences are observed. 1) The N-terminal antiparallel β-sheet of TaNAPRTase is composed of six β-strands instead of the four found in MtQAPRTase by adding extra two β-strands from the C-terminal domain; 2) The starting region of the N terminus is composed of a loop and a short α-helix involved in dimeric interaction, unlike the long N-terminal helix of MtQAPRTase not involved in dimeric interaction. The seven β-stranded α/β barrel structure of the central domain of TaNAPRTase has been first observed in QAPRTase from S. typhimurium (StQAPRTase) known as the type II phosphoribosyltransferase fold (26Eads J.C. Ozturk D. Wexler T.B. Grubmeyer C. Sacchettini J.C. Structure. 1997; 5: 47-58Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). This is a unique fold with an approximate topology of αβ αβ (βα)4β, unlike the conventional topology of (α/β)8. However, some deviations are observed in the TaNAPRTase structure. 1) An extra α-helix H6 is present between β7 and H7; 2) The loop between H6 and H7 does not form a part of the β-barrel due to the large gap with β8. As a result, the actual topology is αβ αβ α2(βα)3β instead of αβ αβ (βα)4β. Active Site Comparison between TaNAPRTase and QAPRTases—NAPRTase and QAPRTase use the same substrate, PRPP, to transfer the phosphoribosyl group and use NA and QA, respectively, to produce NAMN. The comparison of the three active sites of TaNAPRTase and two QAPRTases, StQAPRTase and EcQAPRTase, revealed that the shape and the size of the active site pocket (∼8 × 12 Å) are strongly correlated with the shape and size of the final product, NAMN. The active site pockets are confined by the other dimer subunit. The dimerization is thought to be important in increasing substrate specificity and the proper functioning of TaNAPRTase as shown in the two QAPRTase structures. Except for the dimension of the active sites, other characteristics of the active sites are quite different between TaNAPRTase and the two QAPRTases. The QA-bound structures of the two QAPRTases showed the presence of a strongly positively charged cleft inside the active site pocket to accommodate two carboxylate moieties of QA (24Sharma V. Grubmeyer C. Sacchettini J.C. Structure. 1998; 6: 1587-1599Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, in TaNAPRTase, the active site pocket itself is not so basic due to the presence of a relatively high content of hydrophobic and aromatic residues. The basic residues lining the active site pockets are as follows. 1) StQAPRTase has 8 basic residues, Arg118′ (from the other subunit), Arg152, Lys153, Arg159, Lys163, His174, Arg175, and Lys183; 2) MtQAPRTase has 8 basic residues, Arg105′, Arg136, Arg139, Lys140, His161, Arg162, Lys172, and His274; 3) TaNAPRTase has four basic redues, Arg141, Arg142, His182, and Arg235. Interestingly, there is no conserved residue detected in the active site pockets between TaNAPRTase and the two QAPRTases. Structural Comparison of PRPP-bound Forms—For the transfer of the phosphoribosyl group, PRPP must bind to the enzyme. In type I phosphoribosyltransferases, there are three conserved motifs forming a PRPP-binding site (27Sinha S.C. Smith J.L. Curr. Opin. Struct. Biol. 2001; 11: 733-739Crossref PubMed Scopus (91) Google Scholar). However, TaNAPRTase has a type II phosphoribosyltransferase fold not having these motifs, as first shown in the StQAPRTase structure. The structural comparison was done between PRPP-bound TaNAPRTase and 5-phosphoribosyl-1-(β-methylene) pyrophosphate (a PRPP analog)-bound MtQAPRTase. In both structures, the phosphate group of PRPP binds to a common phosphate-binding motif of many α/β barrel enzymes and QAPRTase (28Wilmanns M Hyde C.C. Davies D.R. Kirschner K. Jansonius J.N. Biochemistry. 1991; 30: 9161-9169Crossref PubMed Scopus (146) Google Scholar). It is known that the phosphate group is bound between the C-terminal ends of the final two β-strands of the α/β barrel, created by residues of the loop between β-strand 7 (β11 in the case of TaNAPRTase) and α-helix 7 (H10) and the N terminus of the additional helix 8′ (H11) of conventional α/β barrel structures (Fig. 6A). Although the sequence similarity among the phosphate-binding motifs is poor, the structures overlay in quite a striking manner. There is almost no conservation of the basic residues that coordinate the phosphate group. The important driving force of phosphate binding is due to main chain amides similar to the oxyanion hole formed by a glycine rich motif as shown in the carboxylesterase structure (29Kim K.K. Song H.K. Shin D.H. Hwang K.Y. Choe S. Yoo O.J. Suh S.W. Structure. 1997; 5: 1571-1584Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Two conserved motifs, 269hSGGh273 (h stands for hydrophobic residue) and 290GVGXX294 (Figs. 2B and 6B), serve to accommodate the phosphate group of PRPP. The comparison of the two complex structures revealed that pyrophosphate moieties are located at the entrance of the active site pocket. However, their specific position and interacting residues are quite different between TaNAPRTase and MtQAPRTase, as shown in Fig. 6B. In the case of MtQAPRTase, although there are few directly interacting residues, several charged residues (Arg48′, Arg105′, Glu104′, Asp173, Glu201, and Asp222) are located to help charged interaction. However, TaNAPRTase has few charged residues, Asp237 and Asp302′, located around PRPP, but Ser240 from the 235RLDTPSSRRG244 motif (Fig. 2B) strongly interacts with PRPP (Fig. 3A). The comparison of native and PRPP complex structures of TaNAPRTase shows no significant conformational changes. As shown in Fig. 3A, only a few side chains lining the active site pocket relocate their positions to avoid steric hindrance with PRPP. Structural Comparison of NAMN-bound Forms—The comparison of NAMN structures of TaNAPRTase and the two QAPRTases shows that NAMNs occupy a different site from PRPP; NAMN is located inside the cavity instead of the entrance (Fig. 6). However, the phosphate group of NAMN is located in approximately the same position as that of PRPP. Therefore, the architecture for accommodating the phosphate moiety of PRPP and NAMN may be critical for enzyme function. The ribosylnicotinate moiety of NAMN swings from outside to inside as compared with the ribosylpyrophosphate moiety of PRPP. To accommodate the two carboxylate groups of QA, StQAPRTase has 5 basic residues (Arg118′, Arg152, Lys153, His174, and Arg175) lining the deep active site pocket (26Eads J.C. Ozturk D. Wexler T.B. Grubmeyer C. Sacchettini J.C. Structure. 1997; 5: 47-58Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). MtQAPRTase has 4 (Arg136, Arg139, His161, and Arg162) in the deep active site pocket to accommodate the QA and NA moiety of NAMN (24Sharma V. Grubmeyer C. Sacchettini J.C. Structure. 1998; 6: 1587-1599Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). TaNAPRTase has only one basic residue, Arg235, in the deep active site. However, to compensate the weak basic potential in the deep active site, TaNAPRTase employs a different strategy to deal with the same product. In addition to specific binding of NAMN to Arg235 from the 235RLDTSSRRG244 motif and hydrogen bond with Thr179 of the 178GTMPH182 motif, the aromatic ring of NA intercalates between the highly conserved aromatic resides Tyr21′ and Phe138 (Figs. 2B and 3B). The distances between these aromatic rings are around 4 Å, the general distance of stacked base pairs in DNA. Therefore, TaNAPRTase uses a stacking effect as a driving force to accommodate NA. C-terminal Unique Zinc Finger Domain—The C-terminal domain (299-393) of TaNAPRTase is composed of seven β-strands followed by the terminal α-helix (H12). The first two β-strands and the last β-strand formed a six-stranded β-sheet with three N-terminal β-strands. The next four β-strands form a zinc knuckle-like structure formed by the 4 cysteines, Cys330, Cys333, Cys348, and Cys350. However, there is no zinc atom in the center of the 4 cysteines. A DALI search (25Holm L. Sander C. Nucleic Acids Res. 1997; 25: 231-234Crossref PubMed Scopus (361) Google Scholar) with the C-terminal domain shows the highest similarity with a DNA replication initiator from Methanobacterium thermoautotrophicum (Z = 2.1; root mean square deviations of 2.7 Å for 39 pairs of aligned Cα atoms; 1ltl-A). Interestingly, the aligned 39 Cα atoms of the DNA replication initiator is composed of a zinc knuckle structure (Fig. 7). This zinc finger domain of the DNA replication initiator is involved in a unique dodecamerization interaction rather than a functional activity (30Fletcher R.J. Bishop B.E. Leon R.P. Sclafani R.A. Ogata C.M. Chen X.S. Nat. Struct. Biol. 2003; 10: 160-167Crossref PubMed Scopus (265) Google Scholar). The PSI-BLAST search with the C-terminal sequence showed that the CXXC(X)14CXC motif is unique and is not conserved in NAPRTase subfamily. Therefore, the role of this motif may be unique to TaNAPRTase. In the tertiary and quaternary structure of TaNAPRTase, this motif does not interact with any other domains, unlike that of the DNA replication initiator. Comparison of TaNAPRTase with Other Homologues Using Sequence Alignment and Secondary Structure Prediction—NAPRTase has been identified in many species from bacteria to human. In the Pfam data base (12Bateman A. Birney E. Durbin R. Eddy S.R. Howe K.L. Sonnhammer E.L. Nucleic Acids Res. 2000; 30: 276-280Crossref Scopus (2021) Google Scholar), TaNAPRTase, St- NAPRTase, EcNAPRTase, and PBEF belong to pfam04095. In the NCBI CDD data base (31Marchler-Bauer A. Anderson J.B. DeWeese-Scott C. Fedorova N.D. Geer L.Y. He S. Hurwitz D.I. Jackson J.D. Jacobs A.R. Lanczycki C.J. Liebert C.A. Liu C. Madej T. Marchler G.H. Mazumder R. Nikolskaya A.N. Panchenko A.R. Rao B.S. Shoemaker B.A. Simonyan V. Song J.S. Thiessen P.A. Vasudevan S. Wang Y. Yamashita R.A. Yin J.J. Bryant S.H. Nucleic Acids Res. 2003; 31: 383-387Crossref PubMed Scopus (650) Google Scholar), they are categorized in different subfamilies. TaNAPRTase belongs to cd01571 (called NAPRTase subgroup B), StNAPRTase belongs to cd01401 (called PncB_like), and PBEF to cd01569 (called PBEF_like). To infer the structural properties of these subfamilies, sequence alignments and secondary structure predictions have been performed and compared with those of TaNAPRTase. As expected, overall structure is predicted to be quite similar (Fig. 2A). In particular, the functionally important type II phosphoribosyltransferase fold is predicted to be present in all the members. The α/β barrel domain is apparent in the secondary structure predictions. NAPRTase has ATPase activity even if it is not essential for a function. StNAPRTase clearly showed the presence of a phosphorylated intermediate after ATP hydrolysis (11Rajavel M. Lalo D. Gross J.W. Grubmeyer C. Biochemistry. 1998; 37: 4181-4188Crossref PubMed Google Scholar). His219 is phosphorylated and converted to phosphohistidine (11Rajavel M. Lalo D. Gross J.W. Grubmeyer C. Biochemistry. 1998; 37: 4181-4188Crossref PubMed Google Scholar). The sequence alignment shows that His219 is not conserved in TaNAPRTase and PBEF subfamilies (Fig. 2A). Therefore, it is not clear whether the formation of a phosphohistidine is the common property of all NAPRTases or not. If this is the case, His182 from the 178GTMPH182 motif of TaNAPRTase is one of the candidates for a phosphorylation site because it is located at the active site pocket. Recently, PBEF from murine has been identified as an NAmPRTase (13Rongvaux A. Shea R.J. Mulks M.H. Gigot D. Urbain J. Leo O. Andris F. Eur. J. Immunol. 2002; 32: 3225-3234Crossref PubMed Scopus (481) Google Scholar). The NAmPRTase function is predicted to originate from a type II phosphoribosyltransferase fold, as discussed above. The sequence alignment shows that the nicotinate-binding residues and phosphate moiety of PRPP-binding motifs are more similar to those of TaNAPRTase. In the NCBI CDD data base, TaNAPRTase (the NAPRTase subgroup B) sequence family is closer to the PBEF sequence family than to the PncB_like sequence family. However, PBEF has a longer N-terminal domain similar to PncB_like and has several insertions not found in other subfamilies. Therefore, the crystal structure of TaNAPRTase together with sequence information show that TaNAPRTase, PBEF, and PncB_like subfamilies have their unique sequence motifs and structural properties for their specific function, although they share a common fold and function. In summary, the first crystal structure of an NAPRTase family member is presented in this study. The presence of the type II phosphoribosyltransferase fold in the TaNAPRTase structure together with bound PRPP and NAMN complex structures helps to explain the NAPRTase function of this family at a molecular level. The application of structural information to analyze subfamilies gives an insight for finding common as well as unique properties of each subfamily. Thus, the crystal structure of TaNAPRTase provides a structural framework for understanding the molecular functions of all members of NAPRTase family. We are grateful to Barbara Gold for cloning, Marlene Henriquez and Bruno Martinez for expression studies and cell paste preparation, and John-Marc Chandonia for bioinformatics search of the gene. We are also grateful to the staff at the Advanced Light Source, which is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract Number DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory." @default.
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• W2080303413 title "Crystal Structure of a Nicotinate Phosphoribosyltransferase from Thermoplasma acidophilum" @default.
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