Insights into Ligand Binding and Catalysis of a Central Step in NAD+ Synthesis

Nicotinamide mononucleotide adenylyltransferase (NMNATase) catalyzes the linking of NMN+ or NaMN+ with ATP, which in all organisms is one of the common step in the synthesis of the ubiquitous coenzyme NAD+, via both de novo and salvage biosynthetic pathways. The structure of Methanobacterium thermoautotrophicum NMNATase determined using multiwavelength anomalous dispersion phasing revealed a nucleotide-binding fold common to nucleotidyltransferase proteins. An NAD+ molecule and a sulfate ion were bound in the active site allowing the identification of residues involved in product binding. In addition, the role of the conserved16HXGH19 active site motif in catalysis was probed by mutagenic, enzymatic and crystallographic techniques, including the characterization of an NMN+/SO 42–complex of mutant H19A NMNATase.1ej21hyb Nicotinamide mononucleotide adenylyltransferase (NMNATase) catalyzes the linking of NMN+ or NaMN+ with ATP, which in all organisms is one of the common step in the synthesis of the ubiquitous coenzyme NAD+, via both de novo and salvage biosynthetic pathways. The structure of Methanobacterium thermoautotrophicum NMNATase determined using multiwavelength anomalous dispersion phasing revealed a nucleotide-binding fold common to nucleotidyltransferase proteins. An NAD+ molecule and a sulfate ion were bound in the active site allowing the identification of residues involved in product binding. In addition, the role of the conserved16HXGH19 active site motif in catalysis was probed by mutagenic, enzymatic and crystallographic techniques, including the characterization of an NMN+/SO 42–complex of mutant H19A NMNATase.1ej21hyb nicotinic acid dinucleotide multiwavelength anomalous dispersion nicotinamide mononucleotide adenylyltransferase wild type glycerol-3-phosphate cytidyltransferase phosphopantetheine adenylyltransferase nicotinic acid mononucleotide root mean square deviation Nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) catalyzes the synthesis of nicotinamide adenine dinucleotide (NAD+) or nicotinic acid dinucleotide (NaAD+)1 from nicotinamide mononucleotide (NMN+) or nicotinic acid mononucleotide (NaMN+), respectively, by transferring the adenylyl part of ATP and concomitantly releasing pyrophosphate (PPi) (Fig. 1 A). The reaction product, NAD+, plays a central role in many cellular processes; it functions as a coenzyme in reduction-oxidation reactions and as a substrate in DNA ligation and protein ADP-ribosylation reactions (1Magni G. Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 135-182PubMed Google Scholar). There is also considerable medical interest in this enzyme as it is implicated in the metabolism of the antitumor drug tiazofurin (2Jayaram H.N. Pillwein K. Lui M.S. Faderan M.A. Weber G. Biochem. Pharmacol. 1986; 35: 587-593Crossref PubMed Scopus (30) Google Scholar). In vivo, tiazofurin is phosphorylated to tiazofurin monophosphate and then converted by NMNATase to the actual pharmacophore thiazole-4-carboxamide adenine dinucleotide, an analog of NAD+. Thiazole-4-carboxamide adenine dinucleotide is a potent inhibitor of inosine monophosphate dehydrogenase causing arrest of guanylate biosynthesis and thus inhibition of tumor cell proliferation. Consistent with this interpretation, low NMNATase activity is observed in cancer patients showing resistance to tiazofurin therapy (2Jayaram H.N. Pillwein K. Lui M.S. Faderan M.A. Weber G. Biochem. Pharmacol. 1986; 35: 587-593Crossref PubMed Scopus (30) Google Scholar). Two mechanisms for the NMNATase-catalyzed synthesis of NAD+have been postulated; the first one assumes a double displacement reaction that involves the formation of an adenylyl enzyme covalent intermediate upon release of pyrophosphate followed by transfer of the adenylyl group to NMN+ to form NAD+, whereas the second one describes a nucleophilic attack of the 5′-phosphate of NMN+ on the α-phosphate of ATP to form NAD+and releasing PPi. The latter mechanism is supported by17O NMR studies of NAD+ synthesis (3Lowe G. Tansley G. Eur. J. Biochem. 1983; 132: 117-120Crossref PubMed Scopus (19) Google Scholar), but a complete understanding of the catalytic chemistry awaited more detailed structural information. NMNATase proteins have been identified, purified, and characterized from archaea, bacteria, and eukarya. All of these proteins are oligomeric; trimeric, tetrameric, and hexameric forms have been observed (1Magni G. Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 135-182PubMed Google Scholar). Several NMNATase genes have been sequenced from a variety of sources (Fig. 1 B). Although these gene products remain annotated in the GenBankTM data base as of unknown function, related sequences from Methanococcus jannaschii, Escherichia coli, Synechocystis sp., and Sulfolobus solfataricus have been overexpressed as recombinant proteins inE. coli and shown to exhibit NMNATase activity (4Raffaelli N. Pisani F.M. Lorenzi T. Emanuelli M. Amici A. Ruggieri S. Magni G. J. Bacteriol. 1997; 179: 7718-7723Crossref PubMed Google Scholar, 5Raffaelli N. Emanuelli M. Pisani F.M. Amici A. Lorenzi T. Ruggieri S. Magni G. Mol. Cell. Biochem. 1999; 193: 99-102Crossref PubMed Google Scholar, 6Raffaelli N. Lorenzi T. Amici A. Emanuelli M. Ruggieri S. Magni G. FEBS Lett. 1999; 444: 222-226Crossref PubMed Scopus (42) Google Scholar, 7Emmanuelli M. Carnevali F. Lorenzi M. Raffaelli N. Amici A. Ruggieri S. Magni G. FEBS Lett. 1999; 455: 13-17Crossref PubMed Scopus (51) Google Scholar, 8Raffaelli N. Lorenzi T. Mariani P.L. Emanuelli M. Amici A. Ruggieri S. Magni G. J. Bacteriol. 1999; 181: 5509-5511Crossref PubMed Google Scholar). Recently, the crystal structure of NMNATase from M. jannaschii in complex with ATP and Mg2+ was reported (9D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar). Our results on the NAD+ and NMN+ complexes of the Methanobacterium thermoautotrophicum enzyme complement this result, especially when interpreting the catalytic mechanism of NMNATase. We describe the crystal structures of the NAD+ complex of NMNATase and of the NMN+complex of an active site mutant (H19A) of NMNATase at 1.9 and 2.5 Å resolution, respectively. These structural results, combined with mutagenesis and enzymatic experiments, define the spatial geometry of the ligand binding sites, identify residues with potential roles in substrate binding and catalysis, and suggest aspects of the product release mechanism. The NMNATase gene (GenBankTM accession number AE000803) was amplified by polymerase chain reaction using M. thermoautotrophicum genomic DNA and cloned into the pET15b (Novagen) expression vector at the NdeI and BglII sites. Recombinant NMNATase was overexpressed in E. coliBL21 Gold (DE3) cells (Stratagene) harboring a plasmid encoding rareE. coli tRNA genes. The cells were grown at 37 °C in Luria-Bertoni broth with carbenicillin (50 μg/ml) and kanamycin (50 μg/ml) to an A 600 nm of 0.7 and induced overnight with 0.5 mmisopropyl-β-d-thiogalactopyranoside at 24 °C. The bacteria were harvested by centrifugation and resuspended in binding buffer (50 mm Tris, 500 mm NaCl, 5% glycerol, and 5 mm imidazole) supplemented with 2 mmphenylmethylsulfonyl fluoride. Bacteria were lysed by several passages through a French pressure cell at 1.4 × 108 pascals, and DNA was sheared by sonication. Cell debris was removed through centrifugation for 30 min at 20,000 × g. ContaminatingE. coli proteins were removed by heating for 20 min at 55 °C followed by centrifugation at 5000 × g for 30 min. The supernatant was applied by gravity to a DE52 column (Whatman) immediately coupled to a Ni2+ column (Qiagen). The Ni2+ column was washed with 50 volumes of binding buffer containing 30 mm imidazole. The bound NMNATase was eluted from the Ni2+ column with 500 mm imidazole in binding buffer. The hexahistidine tag was cleaved by digesting for 16 h with thrombin (1 μg of thrombin per mg of recombinant protein) at 4 °C in binding buffer made 2.5 mm in CaCl2. NMNATase was then dialyzed against 500 mm NaCl in 10 mm HEPES (pH 7.5) and concentrated to 10 mg/ml using BioMax concentrators (Millipore). Mutant H19A NMNATase was purified as described above for WT NMNATase. For the preparation of selenomethionine (Se-Met)-enriched protein, NMNATase was expressed in a methionine auxotroph strain B834(DE3) of E. coli (Novagen) and purified under the same conditions as native NMNATase with the addition of 5 mmβ-mercaptoethanol in all buffers. Screening for crystallization conditions was performed using Hampton Research Crystal Screens I and II at room temperature in VDX plates with the hanging drop vapor diffusion method. 2 μl of protein solution (10 mg/ml) were mixed with 2 μl of the various reservoir solutions and equilibrated with 500 μl of this solution. Crystals in the form of hexagonal rods appeared after 24 h in crystallization set-ups containing ammonium sulfate or lithium sulfate as precipitant. Crystals selected for native and multiwavelength anomalous dispersion (MAD) data collection were grown in 1.5 m LiSO4 and 100 mm HEPES at pH 7.5 at 20 °C. Crystals of H19A NMNATase were grown in 1.6m LiSO4 and 100 mm HEPES at pH 7.5 at 20 °C. Making use of the anomalous scattering of selenium atoms, a three-wavelength MAD experiment was carried out at 100 K on beamline BM14D, APS, using a Q1 CCD detector (Area Detector Systems Corp.). The crystal was flash-frozen with crystallization buffer plus 30% glycerol as cryoprotectant. Diffraction data from native crystals of WT and H19A NMNATase were collected on beamline BM14C, APS, at 100 K using a Q4 CCD detector (ADSC). Both MAD and native data were processed and scaled with the DENZO/SCALEPACK suite of programs (10Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38592) Google Scholar). The selenium sites were located using SOLVE (11Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar) and refined using SHARP (12de la Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar). The electron density map was improved using Density Modification from the CCP4 package (13Bailey S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (42) Google Scholar). Model building was done with O (14Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar), and CNS (crystallography and NMR system) (15Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros J. M. T. Acta Crystallogr. Sect. D Biol. Crystallogr. PubMed Scopus Google Scholar) was for were using CNS and then in O using the a of at 2.5 in an a of at in a and and are in and respectively. The programs J. Crystallogr. 1991; Google Scholar), Acta Crystallogr. Sect. D Biol. Crystallogr. 1991; 50: Scopus Google Scholar), J. A and The for A Scholar), and 8: PubMed Scopus Google Scholar) were in the of the of data collection cell × × × × × × × × × × sites I is the observed and is the from of of of is the and is the and after and after and with Density in to the Å for the native Å for the MAD and Å for the mutant I is the observed and is the from of of is the and is the and after and after and with Density respectively. in a of was using selected in a in to the Å for the native Å for the MAD and Å for the mutant was using selected of NMNATase was performed with a column equilibrated with 10 mm HEPES and 500 using pressure and was performed at 4 °C at a of 0.5 mutagenesis to to was carried out using The were and for DNA encoding WT NMNATase cloned into was as for the polymerase chain reaction mutagenesis of DNA was with the DNA polymerase using the in the the temperature was to reaction and at 37 °C for h followed by of the plasmid into WT NMNATase activity was in a coupled to Raffaelli N. Lorenzi T. Amici A. Emanuelli M. Ruggieri S. Magni G. FEBS Lett. 1999; 444: 222-226Crossref PubMed Scopus (42) Google Scholar). of NMNATase were with 2 2 mm ATP, 10 and 50 mm HEPES (pH 7.5) at °C for 20 min. The of NAD+ was at nm using dehydrogenase to NAD+ to The enzymatic activity of H19A NMNATase was the same but the of enzyme in from 1 to 5000 The structure of the of NMNATase been determined by the MAD using selenium as the anomalous The electron density is of for the of residues in which the chain density is but the of chain In addition, and are in the electron density The with in a one molecule of 1 and 1 sulfate ion (Fig. 2 A). at 1.9 Å in an of and an of to J. Crystallogr. Google Scholar), of the residues are in the and is in the of the The structure of the NMN+ complex of H19A NMNATase was determined by electron density map is of the including residues and for which electron density is observed at The of this complex and with 10 1 molecule of and 1 sulfate at 2.5 Å an of and of of the residues are in the and is in the Consistent with which for M. thermoautotrophicum NMNATase to a as the in solution the of NMNATase crystals the into a hexameric B). is by by and on of with of × × of (Fig. 2 the first of which is the and residues with a of and The at the of the of with (Fig. 2 A). The second is made from and is the to The are and 5 of A and of are by and with and and with These are and as as and A (Fig. 2 B). In of a of of are upon In to the the are and The of and of A against of of D with the same and as as and F. In of a of of are (Fig. 2 B). The active site is located in a a the of the The site is to to release the product NAD+, with a sulfate in the electron density after the first of NAD+ was enzyme and NMNATase have NAD+ in an with adenine an and both the adenylyl and the nicotinamide showing In the adenylyl to in the in the M. jannaschii NMNATase structure (9D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar). The of adenine is to the chain of and the of which to the chain of and to a The adenylyl forms an Å and the of in to been in in which the adenylyl is in by a located at the of the second Struct. Biol. PubMed Scopus Google Scholar). In it is the nicotinamide in which one and the chain of the with the chain of The nicotinamide with the of and to the chain of the to and the to In addition, the chain of the of the in (Fig. the enzyme to both NMN+ and NaMN+ as an chain is for this A an for as as A of the the for the second in the of a in to as this of the chain the of the protein molecule and The in the pyrophosphate forms to The the of the pyrophosphate group the chain of The to the chain of and a electron density that as a ion to the of the pyrophosphate and of the of which is located Å from the of the the and of this a sulfate an of a Both and sulfate have been by the crystallization buffer m NaCl, 1.5 m The above and the ion are located from the and the The sulfate are bound to the of and (Fig. and residues the and (Fig. 1 and also to the group of and the of The with to as the in the M. jannaschii structure is a (9D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar). The of the sulfate ion is with it the binding site of the of the substrate ATP and is of the sulfate ion bound at the in the active site of tRNA with M.A. T.A. PubMed Scopus Google Scholar). by interpreting the the binding of to M. jannaschii NMNATase (9D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar). of the structure of NMNATase using the J. Mol. Biol. PubMed Scopus Google Scholar) several proteins all of to the nucleotidyltransferase of fold containing T. A. J. 1999; PubMed Scopus Google Scholar). of this are cytidyltransferase S. Struct. Des. 1999; Full Text Full Text PDF Scopus Google Scholar), tRNA M.A. D T.A. Scopus Google Scholar), tRNA J. Mol. Biol. PubMed Scopus Google Scholar) and phosphopantetheine adenylyltransferase T. A. J. 1999; PubMed Scopus Google Scholar). structures are of and atoms, to and the fold of NMNATase but forms a as the the of to is All of these related proteins a nucleotide-binding the active site motif and a reaction that is to that of NMNATase. reaction involves the attack of a nucleophilic group of one substrate at the α-phosphate of the second a and releasing a pyrophosphate identification of NAD+ in the active site and the results nucleotidyltransferase to that protein annotated as in thermoautotrophicum data base J. T. K. W. J. N. A. S. S. G. A. S. G. J. J. J. Bacteriol. 1997; 179: PubMed Scopus Google Scholar), was in an NMNATase. enzymatic that the enzyme the biosynthesis of NAD+ from NMN+ and ATP The of this was when a revealed that the enzyme from M. jannaschii NMNATase activity (4Raffaelli N. Pisani F.M. Lorenzi T. Emanuelli M. Amici A. Ruggieri S. Magni G. J. Bacteriol. 1997; 179: 7718-7723Crossref PubMed Google Scholar, 6Raffaelli N. Lorenzi T. Amici A. Emanuelli M. Ruggieri S. Magni G. FEBS Lett. 1999; 444: 222-226Crossref PubMed Scopus (42) Google Scholar). In addition to the common nucleotide-binding all of these an active site that is in all and in M. 1 B). active site motif also been in binding adenylyltransferase reactions as the ATP of A. ATP and E. coli adenine dinucleotide J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. T. K. 2000; PubMed Scopus Google Scholar, G. V. M. J. J. M.A. 1997; PubMed Scopus Google Scholar). For several of these the roles of the residues in this motif have been using crystallographic and mutagenesis M.A. T.A. PubMed Scopus Google Scholar, T. A. J. 1999; PubMed Scopus Google Scholar, S. Struct. Des. 1999; Full Text Full Text PDF Scopus Google Scholar, M.A. D T.A. Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar, S. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Although to residues in and tRNA that were for catalysis J. T. K. W. J. N. A. S. S. G. A. S. G. J. J. J. Bacteriol. 1997; 179: PubMed Scopus Google Scholar, S. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar), structural studies of tRNA were more in the binding of the and of ATP and the of the M.A. T.A. PubMed Scopus Google Scholar). of of the residues and to in and of catalytic in of E. coli cells that the mutant proteins of adenylyltransferase activity J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the binding sites of the are characterized and for all of the nucleotidyltransferase is the one for which the with the product have been described T. A. J. 1999; PubMed Scopus Google Scholar). enzyme is also the one with the of structural to NMNATase. NMNATase and are using it is that the adenylyl but the of the products of the active sites, (Fig. 4 is to that that is by the of A in is by the chain of in through the nicotinamide with the to a the structural motif of tRNA and on that of NMNATase the in from which to the and of ATP (Fig. 4 A). The of in NMNATase activity was using enzymatic that the H19A mutant of the activity of WT a in catalysis for this (Fig. 4 B). The results are also in with studies of and in which mutagenesis of the residues to in with by at 4 of J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Eur. J. Biochem. PubMed Scopus Google Scholar). that the H19A protein was and and that of NMNATase activity was by or crystal structure was The mutant enzyme in the same crystal form as WT structure was to that of WT NMNATase of all is of the product NAD+, a molecule of the substrate in active site (Fig. B). the that a of NMNATase activity for this this a of for ATP and an to the chemistry for The in also to for the structural the protein of the native and mutant In WT the residues and of both the and is density to the For residues in the H19A is electron density at the of the adenine of ATP or NAD+ is to at this of Several and residues and the active site of and with ATP have been The of covalent intermediate also been (9D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar). the of ATP is with the results of the crystal structure of H19A NMNATase with it that bound in a that group is to the α-phosphate of ATP, to attack from the of the pyrophosphate in with attack adenylyltransferase mechanisms M.A. T.A. PubMed Scopus Google Scholar). A in the of the α-phosphate of ATP to the of NAD+. to in a of the of the from to The crystallographic results are with NMR studies of NAD+ that attack of ATP by NMN+ (3Lowe G. Tansley G. Eur. J. Biochem. 1983; 132: 117-120Crossref PubMed Scopus (19) Google Scholar). the group of NMN+ is a and pyrophosphate a residues from the active site of NMNATase to in the chemistry of the reaction by or covalent catalysis M.A. T.A. PubMed Scopus Google Scholar, J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the reaction into the geometry and all that is for catalysis to An adenylyltransferase mechanism was first reported for tRNA by tRNA M.A. T.A. PubMed Scopus Google Scholar). We that this is the mechanism by the and the active site motif is at an all organisms for which the gene been sequenced (Fig. 1 B). The of (9D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar) and that have the to the from whereas an mechanism (1Magni G. Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 135-182PubMed Google Scholar). M. thermoautotrophicum NMNATase is expressed in E. are the active site of the Full catalytic of this enzyme is observed at the temperature of the NAD+ bound to WT NMNATase the protein was to °C These results to an in this protein the release of product the binding of the ATP the the adenine is as by the of electron density in the NMN+ complex of H19A NMNATase. (9D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar) that in M. jannaschii which to in the M. is bound to one of the of the of chain with the adenine (9D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. Fold. Des. 2000; 8: 993-1004Abstract Full Text Full Text PDF Scopus (67) Google Scholar). NAD+ is this to in for chain and density at the of the as in structure of the NMNATase this it is that in the NMNATase complex is from the adenine (Fig. group is Å from it is in the We that at room temperature the is to the chain from the adenine NAD+ bound at the active the from the adenine as the first step in product M. thermoautotrophicum NMNATase is one of a number of proteins from organisms to with when purified from E. S. A. PubMed Scopus Google Scholar, A. A. A. V. V. I. G. N. K. M.A. M. Struct. Biol. 2000; PubMed Scopus Google Scholar). this for more it to the use of proteins in the of to unknown of We the of for data collection at of the V. S. and for structure

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