Mutational, Structural, and Kinetic Studies of the ATP-binding Site of Methanobacterium thermoautotrophicum Nicotinamide Mononucleotide Adenylyltransferase
2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês
10.1074/jbc.m205369200
ISSN1083-351X
Autores Tópico(s)RNA modifications and cancer
ResumoSeveral residues lining the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase (NMNATase) were mutated in an effort to better characterize their roles in substrate binding and catalysis. Residues selected were Arg-11 and Arg-136, both of which had previously been implicated as substrate binding residues, as well as His-16 and His-19, part of the HXGH active site motif and postulated to be of importance in catalysis. Kinetic studies revealed that both Arg-11 and Arg-136 contributed to the binding of the substrate, ATP. When these amino acids were replaced by lysines, the apparent Km values of the respective mutants for ATP decreased by factors of 1.3 and 2.9 and by factors of 1.9 and 8.8 when the same residues were changed to alanines. All four Arg mutants displayed unaltered Km values for NMN. The apparent k cat values of the R11K and R136K mutants were the same as those of WT NMNATase but the apparent k cat values of the alanine mutants had decreased. Crystal structures of the Arg mutants revealed NAD+ and SO42- molecules trapped at their active sites. The binding interactions of NAD+ were unchanged but the binding of SO42- was altered in these mutants compared with wild type. The alanine mutants at positions His-16 and His-19 retained ∼6 and 1.3%, respectively, of WT NMNATase activity indicating that His-19 is a key catalytic group. Surprisingly, this H19A mutant displayed a novel and distinct mode of NAD+ binding when co-crystallized in the presence of NAD+ and SO42- . Several residues lining the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase (NMNATase) were mutated in an effort to better characterize their roles in substrate binding and catalysis. Residues selected were Arg-11 and Arg-136, both of which had previously been implicated as substrate binding residues, as well as His-16 and His-19, part of the HXGH active site motif and postulated to be of importance in catalysis. Kinetic studies revealed that both Arg-11 and Arg-136 contributed to the binding of the substrate, ATP. When these amino acids were replaced by lysines, the apparent Km values of the respective mutants for ATP decreased by factors of 1.3 and 2.9 and by factors of 1.9 and 8.8 when the same residues were changed to alanines. All four Arg mutants displayed unaltered Km values for NMN. The apparent k cat values of the R11K and R136K mutants were the same as those of WT NMNATase but the apparent k cat values of the alanine mutants had decreased. Crystal structures of the Arg mutants revealed NAD+ and SO42- molecules trapped at their active sites. The binding interactions of NAD+ were unchanged but the binding of SO42- was altered in these mutants compared with wild type. The alanine mutants at positions His-16 and His-19 retained ∼6 and 1.3%, respectively, of WT NMNATase activity indicating that His-19 is a key catalytic group. Surprisingly, this H19A mutant displayed a novel and distinct mode of NAD+ binding when co-crystallized in the presence of NAD+ and SO42- . Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase (NMNATase, 1The abbreviations used are: NMNATase, nicotinamide mononucleotide adenylyltransferase; PAPS, adenosine 3′-phosphate 5′-phosphosulfate; NAD+, nicotinamide adenine dinucleotide; NaAD+, nicotinic acid adenine dinucleotide; WT, wild type; GCT, glycerol-3-phosphate cytidyltransferase; NMN+, nicotinamide mononucleotide; NaMN+, nicotinic acid mononucleotide; r.m.s.d., root mean square deviation.1The abbreviations used are: NMNATase, nicotinamide mononucleotide adenylyltransferase; PAPS, adenosine 3′-phosphate 5′-phosphosulfate; NAD+, nicotinamide adenine dinucleotide; NaAD+, nicotinic acid adenine dinucleotide; WT, wild type; GCT, glycerol-3-phosphate cytidyltransferase; NMN+, nicotinamide mononucleotide; NaMN+, nicotinic acid mononucleotide; r.m.s.d., root mean square deviation. EC 2.7.7.1) is a hexameric enzyme, which catalyzes the reversible biosynthesis of both NAD+ and nicotinic acid adenine dinucleotide (NaAD+) from ATP and nicotinamide mononucleotide (NMN+) or nicotinic acid mononucleotide (NaMN+), respectively. NMNATase was first characterized from yeast autolysate (1Kornberg A. J. Biol. Chem. 1948; 176: 1475-1476Abstract Full Text PDF PubMed Google Scholar) and from crude Escherichia coli lysate (2Dahmen W. Webb B. Preiss J. Arch. Biochem. Biophys. 1967; 120: 440-450Crossref PubMed Scopus (28) Google Scholar). More recently, Magni and co-workers analyzed NMNATase from Methanobacterium jannaschii (3Raffaelli N. Pisani F.M. Lorenzi T. Emanuelli M. Amici A. Ruggieri S. Magni G. J. Bacteriol. 1997; 179: 7718-7723Crossref PubMed Google Scholar), Sulfolobus solfataricus (4Raffaelli N. Amici A. Emanuelli M. Ruggieri S. Magni G. FEBS Lett. 1994; 355: 233-236Crossref PubMed Scopus (15) Google Scholar), and Synechocystis sp. (5Raffaelli N. Lorenzi T. Amici A. Emanuelli M. Ruggieri S. Magni G. FEBS Lett. 1999; 444: 222-226Crossref PubMed Scopus (42) Google Scholar). It was assumed that in all these cases a single enzyme catalyzes both reactions, although NaMN+ and NMN+ served as substrates with quite different affinities (3Raffaelli 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. Emanuelli M. Amici A. Ruggieri S. Magni G. Methods Enzymol. 2001; 331: 281-292Crossref PubMed Scopus (9) Google Scholar). Genomic analysis of E. coli revealed, however, that in this organism there are two homologous but distinct genes, NadD and NadR, which encode the enzymes nicotinic acid mononucleotide adenylyltransferase and nicotinamide mononucleotide adenylyltransferase, respectively (7Mehl R.A. Kinsland C. Begley T.P. J. Bacteriol. 2000; 182: 4372-4374Crossref PubMed Scopus (41) 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). Searching the M. thermoautotrophicum proteomic data base for homologues of NadD, however, did not identify any putative nicotinic acid mononucleotide adenylyltransferase. Archaeal proteomes seem to contain only a single protein that catalyzes both activities. All archaeal enzymes contain an invariant glutamine residue at position 84 that binds to the amide nitrogen of the NMN+ substrate via its amide oxygen side chain (9Saridakis 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). A possible model, which would explain the dual enzymatic activity, has free rotation around the glutamine side chain allowing its amide nitrogen to bind to the carboxylate of NaMN+ making it quite feasible for a single enzyme to catalyze both reactions.Recently, there has been increased interest in the biochemistry of NMNATases; the genes encoding these enzymes in several bacterial, archaeal, and eukaryotic sources including humans have been identified (3Raffaelli 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. Lorenzi T. Amici A. Emanuelli M. Ruggieri S. Magni G. FEBS Lett. 1999; 444: 222-226Crossref PubMed Scopus (42) Google Scholar, 6Raffaelli N. Lorenzi T. Emanuelli M. Amici A. Ruggieri S. Magni G. Methods Enzymol. 2001; 331: 281-292Crossref PubMed Scopus (9) Google Scholar, 8Raffaelli N. Lorenzi T. Mariani P.L. Emanuelli M. Amici A. Ruggieri S. Magni G. J. 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Chem. 2001; 276: 406-412Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The structures of M. thermoautotrophicum and M. jannaschii NMNATase were determined in complex with the product NAD+ or the substrate ATP, respectively (9Saridakis 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, 15D'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 (66) Google Scholar). More recently, crystal structures were reported for the apo-form of human NMNATase (16Garavaglia S. D'Angelo I. Emanuelli M. Carnevali F. Pierella F. Magni G. Rizzi M. J. Biol. Chem. 2002; 277: 8524-8530Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and its complexes with NMN+ (17Werner E. Ziegler M. Lerner F. Schweiger M. Heinemann U. FEBS Lett. 2002; 516: 239-244Crossref PubMed Scopus (35) Google Scholar), NAD+ (18Zhou T. Kurnasov O. Tomchick D.R. Derk B.D. Grishin N.V. Marquez V.E. Osterman A.L. Zhang H. J. Biol. Chem. 2002; 11: 13148-13154Abstract Full Text Full Text PDF Scopus (79) Google Scholar), NaAD+ (18Zhou T. Kurnasov O. Tomchick D.R. Derk B.D. Grishin N.V. Marquez V.E. Osterman A.L. Zhang H. J. Biol. Chem. 2002; 11: 13148-13154Abstract Full Text Full Text PDF Scopus (79) Google Scholar), and the oncolytic agent tiazofurin (18Zhou T. Kurnasov O. Tomchick D.R. Derk B.D. Grishin N.V. Marquez V.E. Osterman A.L. Zhang H. J. Biol. Chem. 2002; 11: 13148-13154Abstract Full Text Full Text PDF Scopus (79) Google Scholar). All these analyses revealed a hexameric assembly and a highly similar overall fold. The structures of Bacillus subtilis and E. coli nicotinic acid mononucleotide adenylyltransferase (the products of the NadD gene) were also determined (19Olland A.M. Underwood K.W. Czerwinski R.M. Lo M.C. Aulabaugh A. Bard J. Stahl M.L. Somers W.S. Sullivan F.X. Chopra R. J. Biol. Chem. 2002; 277: 3698-3707Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 20Zhang H. Zhou T. Kurnasov O. Cheek S. Grishin N.V. Osterman A. Struct. Fold Des. 2002; 10: 69-79Abstract Full Text Full Text PDF Scopus (63) Google Scholar). In contrast to the archaeal and human NMNATases, these bacterial enzymes form dimers or monomers, respectively. Their overall fold, however, is again quite similar.NMNATase adopts a modified Rossmann-fold with features that place the enzyme in the nucleotidyltransferase superfamily. Other members of this protein family include class I tRNA synthetases, ATP sulfurylase, adenosine 3′-phosphate 5′-phosphosulfate (PAPS) synthase, phosphopantetheine adenylyl-transferase, CTP-phosphocholine cytidylyltransferase, glycerol-3-phosphate cytidylyltransferase (GCT), and pantothenate synthetase (21Eriani G. Delarue M. Poch O. Gangloff J. Moras D. 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Besides the nucleotide-binding fold, their common features are a T/HXGH sequence motif at their active sites, and the catalysis of α,β-phosphodiester bond cleavage of either ATP or CTP leading to mononucleotide transfer. The importance of the T/HXGH motif can be demonstrated by its absolute conservation in all the proteins that belong to the nucleotidyltransferase superfamily. In a number of members of this enzyme family, including NMNATase, tyrosyl-tRNA synthetase, PAPS synthase, and GCT, the T/HXGH active site sequence motif has been further characterized using structural and mutational techniques (9Saridakis 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, 15D'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 (66) Google Scholar, 26Veitch D.P. Gilham D. Cornell R.B. Eur. J. Biochem. 1998; 255: 227-234Crossref PubMed Scopus (46) Google Scholar, 28Perona J.J. Rould M.A. Steitz T.A. Biochemistry. 1993; 32: 8758-8771Crossref PubMed Scopus (182) Google Scholar, 29Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (91) Google Scholar). The crystal structures of all of these proteins, with the exception of PAPS synthase, have been determined in the presence of their ATP or CTP substrate. In two of three cases the side chain of the first amino acid of the T/HXGH motif was found to interact with the β-phosphate of ATP (bond distances of 3.1 Å for B. subtilis GCT and 2.8 Å for M. jannaschii NMNATase). The second histidine was bound to the α-phosphate of the triphosphate (bond distances of 3.5 Å for B. subtilis GCT and 3.2 Å for M. jannaschii NMNATase) (15D'Angelo I. Raffaelli N. Dabusti V. Lorenzi T. Magni G. Rizzi M. Struct. 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Green and Co., London1930Google Scholar) and Pauling (32Pauling L. Chem. Eng. News. 1946; 24: 1375-1377Crossref Scopus (718) Google Scholar).An energy-minimized molecule of ATP was modeled into the active site of yeast ATP sulfurylase based on the crystal structure of the protein complexed with product, adenosine 5′-phosphosulfate (23Ullrich T.C. Blaesse M. Huber R. EMBO J. 2001; 20: 316-329Crossref PubMed Scopus (87) Google Scholar). The authors proposed a novel mode of ATP binding in which the T/HXGH motif interacts with the β- and γ-phosphates rather than the α- and β-phosphates of ATP as had been found with the other family members (15D'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 (66) Google Scholar, 29Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (91) Google Scholar).Given these discrepancies in the way triphosphate binding is envisaged in the various family members, we have applied mutational, structural, and kinetic techniques to further characterize the roles of several active site residues proposed to be important in substrate binding and/or catalysis of NMNATase.MATERIALS AND METHODSSite-directed Mutagenesis, Protein Purification, and Crystallization—Site-directed mutagenesis, protein expression, purification, and crystallization were performed as previously described with some minor modifications, mainly involving cell lysis, which now consisted of 5 rounds of sonication (1 min at 50% of maximum output, Branson sonifier 450) (9Saridakis 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). Diffraction data were collected from crystals grown in 1.5–1.6 m (NH4)2SO4, 5% glycerol, and 100 mm Tris-HCl, pH 8.0, at 20 °C and flash-frozen with crystallization buffer plus 30% glycerol as cryoprotectant. To obtain co-crystals of the NAD+ complex of the H19A mutant of NMNATase, the protein (10 mg/ml) was incubated at 65 °C with 5 mm NAD+ for 5 min to release any trapped nucleotide and replace it by NAD+. The Ammonium Sulfate Grid Screen™ (Hampton Research) performed at room temperature in VDX plates applying the hanging drop vapor diffusion method produced diffraction quality crystals under all of the conditions above pH 6. Crystals chosen for data collection were grown in 1.6 m (NH4)2SO4, 5% glycerol and 100 mm Tris-HCl, pH 8.0, at 20 °C. These crystals were also flash-frozen in crystallization buffer supplemented with 30% glycerol as cryoprotectant.Steady State Kinetic Assays—NMNATase was incubated in a solution containing 5 mm MgCl2, 1% ethanol, 1 unit of alcohol dehydrogenase, and 50 mm HEPES buffer, pH 7.5, at 65 °C for 2 min with either varying amounts of NMN+ or ATP while keeping the concentration of the second substrate at 5 mm. The amount of NAD+ formed was measured spectrophotometrically at 340 nm, using alcohol dehydrogenase to convert NAD+ to NADH. The assays with WT, R11K, and R136K NMNATase utilized 2 μg of enzyme, whereas R11A, H16A, H19A, and R136A required 20 μg of enzyme to obtain significant readings. The assays were linear with time and NMNATase concentration. Approximately 5% of the limiting substrate was converted to product.Kinetic Data Analysis—Velocity data obtained by varying one of the substrate concentrations (either ATP or NMN+ using a 1 × 6 matrix) from three independent measurements were averaged and analyzed using Hypero from Cleland's programs resulting in values for apparent velocity (k cat), the apparent Michaelis constant (Km) as well as the apparent first-order rate constant (k cat/Km) and the resulting errors are S.E. (44Cleland W.W. Methods Enzymol. 1979; 63: 103-138Crossref PubMed Scopus (1925) Google Scholar).X-ray Data collection and Structure Determination—Diffraction data sets from crystals of NMNATase mutants R11A and R11K were collected at a wavelength of 1.10 Å on beamline X8C (National Synchrotron Light Source, Brookhaven National Laboratory) at 100 K using a Q4 CCD detector (ADSC). Diffraction data sets from a crystal of the R136A mutant were measured at a wavelength of 1.00 Å and at 100 K on beamline BM14C (BioCARS, Advanced Photon Source, Argonne National Laboratory) equipped with a Q4 CCD detector (ADSC). Diffraction data from a crystal of the H19A mutant were recorded on a Rigaku FR-C rotating copper anode equipped with MSC-Yale double mirror optics and a MAR research 345 imaging plate area detector. All x-ray data were processed and scaled with the help of the DENZO/SCALEPACK suite of programs (33Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38355) Google Scholar). The H19A mutant structure was determined employing the molecular replacement program package AMoRe (34Navaza J. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 1367-1372Crossref PubMed Scopus (658) Google Scholar, 35Bailey S. Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar). All other crystals were sufficiently isomorphous with the WT NMNATase to allow the immediate use of the RIGID routine of CNS, version 0.9 (36Brunger 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. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar), to correctly place the search model. Model visualization and rebuilding were done with the program O (37Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13003) Google Scholar) and CNS, version 0.9, was used for refinement. Water molecules were initially picked using CNS and then manually verified in O using the following criteria: a peak of at least 2.5 σ in an Fo – Fc map, a peak of at least 1.0 σ in a 2Fo – Fc map, and reasonable intermolecular interactions. Crystallographic and refinement statistics are found in Tables I and II. The programs MOLSCRIPT (38Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), RASTER 3D (39Merrit E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1991; 50: 869-873Crossref Scopus (2854) Google Scholar), and SPOCK (40Christopher, J. A. (1998) SPOCK (Structural Properties Observation and Calculation Kit), The Center for Macromolecular Design, College Station, TXGoogle Scholar) were used in the production of the figures.Table ISummary of x-ray data collection statisticsX-ray dataR11AaX-ray data collected at X8C, NSLS, BNL.R11KaX-ray data collected at X8C, NSLS, BNL.R136AbX-ray data collected at 14BMC, BioCARS, APS, ANL.H19AcX-ray data collected on home source.Space groupP6322P6322P6322P3121Unit cell (Å3)88.3 × 88.3 × 110.488.8 × 88.8 × 109.891.4 × 91.4 × 107.7124.5 × 124.5 × 111.8Resolution (Å)30.0-2.4030.0-2.030.0-2.4015.0-3.00Wavelength (Å)1.1001.1001.0001.5418Total observations (No.)42,952187,240185,814223,204Unique reflections (No.)9,71517,83710,08520,207Intensity (I/σ )14.3 (2.5)25 (6.7)41 (12)20.7 (6.5)Completeness (%)92.9 (96.9)99.4 (100.0)99.8 (100.0)100.0 (100.0)R sym0.083 (0.376)0.081 (0.381)0.077 (0.270)0.140 (0.393)a X-ray data collected at X8C, NSLS, BNL.b X-ray data collected at 14BMC, BioCARS, APS, ANL.c X-ray data collected on home source. Open table in a new tab Table IIRefinement statisticsR11AR11KR136AH19AR cryst19.421.622.519.4R free24.625.426.523.3Protein atoms (#)1335133913354050Solvent atoms (#)9817849Ligand atoms (#)444449147R.m.s.d. bond length (Å)0.0060.0060.0060.007R.m.s.d. bond angle (∘)1.21.21.21.3R.m.s.d. dihedral angle (∘)21.721.221.422.0Average atomic thermal factor protein (Å2)A: 26.635.030.535.9B: 37.4C: 21.4Average atomic thermal factor NAD+ (Å2)A: 34.837.534.439.9B: 47.6C: 28.2Average atomic thermal factor SO24 (Å2)A: 38.239.7B: 44.7C: 30.5 Open table in a new tab RESULTS AND DISCUSSIONSite-directed MutagenesisUsing the crystal structure of M. thermoautotrophicum NMNATase complexed with NAD+ and a sulfate ion as a guide, target residues were chosen for site-directed mutagenesis aiming to investigate the roles of conserved active site arginine and histidine residues in binding and catalysis of M. thermoautotrophicum NMNATase. The selected residues, Arg-11, His-16, His-19, and Arg-136 are completely conserved in all sequenced archaeal NMNATases.Steady-state Kinetic AnalysisThe purified WT and mutant enzymes were analyzed by steady-state kinetics. The Michaelis-Menten parameters were measured using the forward NMNATase reaction (synthesis of NAD+). Both apparent k cat and apparent Km values were determined using Cleland's programs (44Cleland W.W. Methods Enzymol. 1979; 63: 103-138Crossref PubMed Scopus (1925) Google Scholar). Table III shows the results of linear regression analyses of data collected from activity assays using each mutant enzyme as well as WT. All of the kinetic values are apparent values.Table IIIMichaelis-Menten parameters of WT and mutant NMNATaseProteinATPNMNkcatKmk cat/KmKmk cat/Kms-1mmmm-1 s-1mmmm-1 s-1WT10 ± 10.16 ± 0.0263 ± 20.08 ± 0.02125 ± 20R11A0.5 ± 0.10.33 ± 0.051.7 ± 0.20.14 ± 0.023.6 ± 0.3R11K2.9 ± 0.10.21 ± 0.0214 ± 10.13 ± 0.0222 ± 2R136A0.4 ± 0.11.4 ± 0.30.3 ± 0.10.11 ± 0.024.0 ± 0.1R136K8 ± 10.46 ± 0.0217 ± 40.14 ± 0.0257 ± 6H16A0.6 ± 0.11.1 ± 0.30.5 ± 0.10.20 ± 0.043.0 ± 0.5H19A0.13 ± 0.010.08 ± 0.021.6 ± 0.20.09 ± 0.021.4 ± 0.2 Open table in a new tab Probing the ATP-Phosphate Binding SiteR11K and R136K Mutants—To be able to evaluate the role of charge as compared with size and shape of a given side chain, the two arginine residues implicated in the binding of the γ-phosphate of ATP were individually mutated to lysines. The highly conservative nature of these changes is reflected in the fact that in bacterial NMNATases the position corresponding to Arg-11 is occupied by a lysine (8Raffaelli N. Lorenzi T. Mariani P.L. Emanuelli M. Amici A. Ruggieri S. Magni G. J. Bacteriol. 1999; 181: 5509-5511Crossref PubMed Google Scholar). The structures of M. thermoautotrophicum and M. jannaschii NMNATase reveal that both Arg-11 and Arg-136 form two interactions each to the γ-phosphate of ATP (or a sulfate molecule occupying its binding site) with hydrogen bond distances varying between 2.8 and 3.2 Å (9Saridakis 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, 15D'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 (66) Google Scholar). In M. thermoautotrophicum NMNATase, Arg-11 also forms a relatively long interaction (3.5-Å interatomic distance) with the "NMN+-phosphate." Both arginine to lysine mutants displayed similar kinetic properties to WT NMNATase with no significant change in k cat for R136K and only a 3.4-fold decrease for R11K. The relatively small changes in affinities for ATP are reflected in Km values of 0.21 and 0.46 mm for R11K and R136K, respectively. For comparison, the Km for ATP of WT NMNATase is 0.16 mm. There is an approximate 4-fold decrease in k cat/Km for ATP for both arginine to lysine mutants, caused by a combination of moderate differences in both k cat and Km values. Overall, lysine can replace each of Arg-11 and Arg-136 without causing major changes in the kinetic properties of NMNATase. We take this as an indication that at these positions the positive charge of the side chain is most important.The Km values for NMN+ of both mutants were similar to that of WT NMNATase, 0.13 and 0.14 mm for R11K and R136K, respectively, compared with 0.08 mm for WT. Arg-136 does not interact with NMN+; Arg-11, however, forms a long hydrogen bond (3.5 Å) to one of the oxygen atoms of the NMN+-phosphate. It is likely that this interaction contributes to catalysis by aiding in the positioning of the NMN+-phosphate but does not contribute strongly to the binding of NMN+ as reflected in the Km value. Together with a 4-fold decrease of k cat for R11K, this could indicate a minor role for Arg-11 in preparing the NMN+-phosphate for its in-line attack on the α-phosphate of ATP. The shorter length of the lysine side chain and the removal of the bi-dentate potential of an arginine head group could lead to a loss of this support function and might be reflected in the lower k cat of the mutant.Overall, the kinetic results are consistent with the findings of the crystal structure of NMNATase, in which Arg-11 and Arg-136 are directly involved in binding the γ-phosphate of ATP, the part of this phosphate chain of the substrate that is furthest from the point where the actual chemical reaction happens. Therefore, one would expect to see the most pronounced change in parameters describing ATP binding and only minor effects on catalytic roles.R11A and R136A Mutants—Given the very modest changes seen upon replacing Arg-11 and Arg-136 with lysine, more drastic modifications were introduced by constructing the corresponding alanine mutants. ATP binding to NMNATase was now affected more strongly with changes in both the k cat and the Km values for ATP. For R11A, we measured a 20-fold reduced k cat value of 0.5 s–1 compared with 10 s–1 for WT. The increase in the Km value for ATP was 2-fold to 0.30 versus 0.16 mm for WT. The k cat/Km value decreased 37-fold to 1.7 mm–1 s–1 compared with 63 mm–1 s–1 for WT. In the case of the R136A mutant, a k cat value of 0.4 s–1 was measured, a 25-fold decrease when compared with 10 s–1 for WT. The Km value increased 9-fold to 1.4 mm from 0.16 mm for WT resulting in a k cat/Km value of 0.3 mm–1 s–1, a 210-fold decrease compared with 63 s–1 for WT. The much higher Michaelis-Menten constants seen in both mutants reflect the relative contributions of these arginine residues to the binding of ATP by NMNATase. Although the catalytic rates were somewhat reduced for both mutants, it is clear that these positively charged amino acids support triphosphate binding but are not absolutely essential for NMNATase catalysis.H16A and H19A Mutants of the "Fingerprint" Motif—The roles of the two His residues within the T/HXGH active site sequence motif have been well characterized for several members of the nucleotidyltransferase superfamily. In GCT the motif is 14HWGH
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