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• W2106722820 abstract "Phosphoribosylaminoimidazole-succinocarboxamide synthetase (SAICAR synthetase) converts 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) to 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide (SAICAR). The enzyme is a target of natural products that impair cell growth. Reported here are the crystal structures of the ADP and the ADP·CAIR complexes of SAICAR synthetase from Escherichia coli, the latter being the first instance of a CAIR-ligated SAICAR synthetase. ADP and CAIR bind to the active site in association with three Mg2+, two of which coordinate the same oxygen atom of the 4-carboxyl group of CAIR; whereas, the third coordinates the α- and β-phosphoryl groups of ADP. The ADP·CAIR complex is the basis for a transition state model of a phosphoryl transfer reaction involving CAIR and ATP, but also supports an alternative chemical pathway in which the nucleophilic attack of l-aspartate precedes the phosphoryl transfer reaction. The polypeptide fold for residues 204–221 of the E. coli structure differs significantly from those of the ligand-free SAICAR synthetase from Thermatoga maritima and the adenine nucleotide complexes of the synthetase from Saccharomyces cerevisiae. Conformational differences between the E. coli, T. maritima, and yeast synthetases suggest the possibility of selective inhibition of de novo purine nucleotide biosynthesis in microbial organisms. Phosphoribosylaminoimidazole-succinocarboxamide synthetase (SAICAR synthetase) converts 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) to 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide (SAICAR). The enzyme is a target of natural products that impair cell growth. Reported here are the crystal structures of the ADP and the ADP·CAIR complexes of SAICAR synthetase from Escherichia coli, the latter being the first instance of a CAIR-ligated SAICAR synthetase. ADP and CAIR bind to the active site in association with three Mg2+, two of which coordinate the same oxygen atom of the 4-carboxyl group of CAIR; whereas, the third coordinates the α- and β-phosphoryl groups of ADP. The ADP·CAIR complex is the basis for a transition state model of a phosphoryl transfer reaction involving CAIR and ATP, but also supports an alternative chemical pathway in which the nucleophilic attack of l-aspartate precedes the phosphoryl transfer reaction. The polypeptide fold for residues 204–221 of the E. coli structure differs significantly from those of the ligand-free SAICAR synthetase from Thermatoga maritima and the adenine nucleotide complexes of the synthetase from Saccharomyces cerevisiae. Conformational differences between the E. coli, T. maritima, and yeast synthetases suggest the possibility of selective inhibition of de novo purine nucleotide biosynthesis in microbial organisms. Phosphoribosylaminoimidazole-succinocarboxamide synthetase (EC 6.3.2.6, 5′-phosphoribosyl-4-carboxy-5-aminoimidazole:l-aspartate ligase (ADP)) (SAICAR synthetase) 2The abbreviations used are: SAICAR, 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide; CAIR, 4-carboxy-5-aminoimidazole ribonucleotide; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AIR, 5-aminoimidazole ribonucleotide; eSS, Escherichia coli SAICAR synthetase; tSS, Thermatoga maritima SAICAR synthetase; ySS, Saccharomyces cerevisiae SAICAR synthetase. 2The abbreviations used are: SAICAR, 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide; CAIR, 4-carboxy-5-aminoimidazole ribonucleotide; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AIR, 5-aminoimidazole ribonucleotide; eSS, Escherichia coli SAICAR synthetase; tSS, Thermatoga maritima SAICAR synthetase; ySS, Saccharomyces cerevisiae SAICAR synthetase. catalyzes the eighth step in bacterial de novo purine nucleotide biosynthesis, ATP + l-aspartate + CAIR → ADP + Pi + SAICAR. Lukens and Buchanan (1Lukens L.N. Buchanan J.M. J. Biol. Chem. 1959; 234: 1791-1798Abstract Full Text PDF PubMed Google Scholar) first described the enzyme in 1959. In 1962 Miller and Buchanan (2Miller R.W. Buchanan J.M. J. Biol. Chem. 1962; 237: 485-490Abstract Full Text PDF PubMed Google Scholar) demonstrated its presence in a variety of life forms and reported the purification and properties of the synthetase from chicken liver. More recently, the Stubbe laboratory (3Meyer E. Leonard N.J. Bhat B. Stubbe J. Smith J.M. Biochemistry. 1992; 31: 5022-5032Crossref PubMed Scopus (64) Google Scholar) purified SAICAR synthetase from Escherichia coli. The E. coli enzyme exhibits a rapid equilibrium random kinetic mechanism (4Nelson S.W. Binkowski D.J. Honzatko R.B. Fromm H.J. Biochemistry. 2005; 44: 766-774Crossref PubMed Scopus (17) Google Scholar). SAICAR synthetase from Saccharomyces cerevisiae is a monomer (5Levdikov V.M. Grebenko A.I. Barynin V.V. Melik-Adamyan W.R. Lamzin V.S. Wilson K.S. Crystallogr. Rep. 1996; 41: 275-286Google Scholar, 6Levdikov V.M. Barynin V.V. Grebenko A.I. Melik-Adamyan W.R. Lamzin V.S. Wilson K.S. Structure. 1998; 6: 363-376Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 7Antonyuk S.V. Grebenko A.I. Levdikov V.M. Urusova D.V. Melik-Adamyan V.R. Lamzin V.S. Wilson K.S. Crystallogr. Rep. 2001; 46: 687-691Crossref Scopus (7) Google Scholar, 8Urusova D.V. Antonyuk S.V. Grebenko A.I. Lamzin V.S. Melik-Adamyan V.R. Crystallogr. Rep. 2003; 48: 763-767Crossref Scopus (6) Google Scholar) and that from Thermatoga maritima a dimer (9Zhang R. Skarina T. Evdokimova E. Edwards A. Savchenko A. Laskowski R. Cuff M.E. Joachimiak A. Acta Crystallograph. Sect. F Struct. Biol. Cryst. Commun. 2006; 62: 335-339Crossref PubMed Scopus (16) Google Scholar). Comparable enzymes from vertebrates have masses in excess of 330 kDa and possess 6–8 identical subunits of 47 kDa (10Patey C.A. Shaw G. Biochem. J. 1973; 135: 543-545Crossref PubMed Scopus (28) Google Scholar, 11Firestine S.M. Davisson V.J. Biochemistry. 1994; 33: 11917-11926Crossref PubMed Scopus (36) Google Scholar). The vertebrate systems are bifunctional, combining 5-aminoimidazole ribonucleotide carboxylase (AIR carboxylase) and SAICAR synthetase activities (10Patey C.A. Shaw G. Biochem. J. 1973; 135: 543-545Crossref PubMed Scopus (28) Google Scholar, 11Firestine S.M. Davisson V.J. Biochemistry. 1994; 33: 11917-11926Crossref PubMed Scopus (36) Google Scholar, 12Chen Z.D. Dixon J.E. Zalkin H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3097-3101Crossref PubMed Scopus (41) Google Scholar).l-Alanosine can replace l-aspartate as a substrate both in vitro and in vivo for SAICAR synthetase (4Nelson S.W. Binkowski D.J. Honzatko R.B. Fromm H.J. Biochemistry. 2005; 44: 766-774Crossref PubMed Scopus (17) Google Scholar, 13Tyagi A.K. Cooney D.A. Cancer Res. 1980; 40: 4390-4397PubMed Google Scholar, 14Alenin V.V. Ostanin K.V. Kostikova T.R. Domkin V.D. Zubova V.A. Smirnov M.N. Biokhimiya. 1992; 57: 845-855PubMed Google Scholar). The product of the SAICAR synthetase reaction, l-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide, is a potent inhibitor of adenylosuccinate synthetase and adenylosuccinate lyase, being the compound responsible for l-alanosine toxicity (13Tyagi A.K. Cooney D.A. Cancer Res. 1980; 40: 4390-4397PubMed Google Scholar). Many cancers (∼30% of all T-cell acute lymphocytic leukemia, for instance) lack a salvage pathway for adenine nucleotides and rely entirely on de novo biosynthesis (15Batova A. Diccianni M.B. Omura-Minamisawa M. Yu J. Carrera C.J. Bridgeman L.J. Kung F.H. Pullen J. Amylon M.D. Yu A.L. Cancer Res. 1999; 59: 1492-1497PubMed Google Scholar). l-Alanosine is toxic to cell lines of such cancers at concentrations well below those that poison cells with intact salvage pathways. Hence, l-alanosine may be effective as a chemotherapeutic agent in combination with other drugs (15Batova A. Diccianni M.B. Omura-Minamisawa M. Yu J. Carrera C.J. Bridgeman L.J. Kung F.H. Pullen J. Amylon M.D. Yu A.L. Cancer Res. 1999; 59: 1492-1497PubMed Google Scholar).Differences in subunit size, function, and assembly of microbial and vertebrate SAICAR synthetases suggest the potential for selective inhibition of SAICAR synthetases and, hence, the possibility of new antibiotics. Efforts to further develop specific inhibitors of microbial SAICAR synthetases would benefit from a basic understanding of structure-function relations; however, for SAICAR synthetase such information is lacking. To this end, we report the structures of the ADP and ADP·CAIR complexes of E. coli SAICAR synthetase (hereafter, eSS). The latter complex is the first structure of a CAIR-bound SAICAR synthetase and reveals a previously unsuspected requirement for Mg2+ in the recognition of CAIR by the synthetase. The CAIR·ADP complex is consistent with a chemical mechanism composed of two partial reactions, a phosphoryl transfer from ATP and a nucleophilic attack by l-aspartate, but the relative order of the two reactions is unclear. Moreover, the conformation of eSS differs significantly from that of ligand-free SAICAR synthetase from T. maritima in the region of the CAIR binding site, suggesting the possibility of substrate-induced conformational changes in microbial synthetases.EXPERIMENTAL PROCEDURESMaterials—ATP, l-aspartate, NADH, phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase were purchased from Sigma. CAIR was synthesized as described previously (4Nelson S.W. Binkowski D.J. Honzatko R.B. Fromm H.J. Biochemistry. 2005; 44: 766-774Crossref PubMed Scopus (17) Google Scholar). E. coli strain BL21(DE3) came from Invitrogen.Enzyme Preparation—Selenomethionine substitution in eSS employed the inhibition of methionine biosynthesis coupled with selenomethionine supplementation (16Van Duyne G.D. Standaert R.F. Karplus P.A. Schreiber S.L. Clardy J. J. Mol. Biol. 1993; 229: 105-124Crossref PubMed Scopus (1076) Google Scholar). BL21(DE3) cells were transformed with a pET 28b vector containing the eSS insert with an N-terminal His6 tag (4Nelson S.W. Binkowski D.J. Honzatko R.B. Fromm H.J. Biochemistry. 2005; 44: 766-774Crossref PubMed Scopus (17) Google Scholar). All bacterial cultures contained 30 μg/ml kanamycin sulfate (Invitrogen). An overnight culture was prepared in LB media (Sigma), and the cells were isolated by centrifugation (1500 × g for 10 min). The pellet was re-suspended in 24 ml of M9 media, supplemented with 1 mm MgSO4, 0.3 mm FeSO4, and 0.5 μm thiamin. Four ml of inoculant culture was added to each flask containing 650 ml of supplemented M9 media. The flasks were shaken at 37 °C to an A600 of 0.8. The temperature was adjusted to 16 °C, and 35 mg each of l-leucine, l-isoleucine, and l-valine, and 65 mg each of l-phenylalanine, l-lysine, and l-threonine were added as solids to each flask. After shaking for 20 min, 2 ml of a 20 mg/ml solution of l-selenomethionine was added to each flask. Isopropyl β-d-thiogalactopyranoside was added to a final concentration of 0.5 mm after an additional 15 min of agitation. Cells were isolated after 18 h by centrifugation (1500 × g, 10 min), re-suspended in 10 mm KPi (pH 7.0), centrifuged again, and finally re-suspended in 100 ml of lysis buffer containing 50 mm KPi, 300 mm NaCl, and 10 mm imidazole (pH 8.0). Cells were disrupted by sonication in the presence of 0.25 mg/ml lysozyme, 50 μg/ml DNase I, 1 ml of 100 mm phenylmethanesulfonyl fluoride in isopropyl alcohol, and 5 μg/ml leupeptin. The lysate was centrifuged (33,000 × g, 1 h) and the supernatant fluid loaded onto 25 ml of nickel-nitrilotriacetic acid-agarose (Novagen), pre-equilibrated in lysis buffer. The column was washed sequentially with 2 column volumes each of lysis buffer, lysis buffer containing 20 mm imidazole, and lysis buffer containing 40 mm imidazole. eSS was subsequently eluted from the column with lysis buffer containing 250 mm imidazole. Immediately upon elution, dithiothreitol and EDTA were added to the fractions to final concentrations of 5 and 10 mm, respectively. Fractions were pooled and dialyzed overnight in buffer containing 15 mm Tris·HCl, 25 mm KCl, 5 mm MgCl2, 5 mm dithiothreitol, and 5 mm EDTA (pH 8.0). Native protein was prepared using an identical protocol, except cell growth and expression was done in LB media without amino acid supplements.Protein concentration was determined by the method of Bradford (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213177) Google Scholar) using bovine serum albumin as a standard. Protein purity was confirmed by SDS-PAGE (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205955) Google Scholar). Mass determinations of purified protein were done by the Iowa State University core facility using an Applied Biosystems Voyager System 6075 matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The specific activity of eSS was determined using previously described assay conditions (4Nelson S.W. Binkowski D.J. Honzatko R.B. Fromm H.J. Biochemistry. 2005; 44: 766-774Crossref PubMed Scopus (17) Google Scholar). The dependence of velocity on the concentration of Mg2+ was investigated using saturating substrate concentrations (300 μm ATP, 65 μm CAIR, and 7.5 mm l-aspartate), with concentrations of free Mg2+ ranging from 90 to 7000 μm.Crystallization—Crystals were grown by the method of hanging-drop vapor diffusion in VDX plates (Hampton Research). Two μl of protein solution (15 mg/ml protein, 15 mm Tris·HCl, 25 mm KCl, 55 mm MgCl2, 50 mm ADP, 5 mm dithiothreitol, and 5 mm EDTA, pH 8.0) were mixed with 2 μl of well solution (3.4–3.8 m sodium formate and 50 mm Tris·HCl, pH 8.5) and allowed to equilibrate against 0.5 ml of well solution. Crystallization experiments for the ADP complex and CAIR·ADP complex employed selenomethionine-substituted and native proteins, respectively.Data Collection—For the ADP complex, crystals were transferred to a cryoprotectant solution containing 4 m sodium formate, 50 mm Tris·HCl (pH 8.5), 25 mm MgCl2, 25 mm ADP, and 10% (w/v) sucrose. This buffer was supplemented with 1 mm CAIR and 10 mm l-aspartate for the CAIR·ADP complex. After ∼30 s of equilibration, crystals were plunged into liquid nitrogen.For the ADP complex, MAD data were collected on Beamline 4.2.2 of the Advanced Light Source, Lawrence Berkley Laboratory. Complete anomalous sets were taken at wavelengths of peak absorbance, the inflection point, and remote from the absorption edge of selenium. Data were indexed, integrated, scaled, and merged using d*trek (19Pflugrath J.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1414) Google Scholar). Intensities were converted to structure factors using the CCP4 (20Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19698) Google Scholar) program TRUNCATE.Data from the CAIR·ADP complex were collected at Iowa State University from a single crystal (temperature, 115 K) on a Rigaku R-AXIS IV++ rotating anode/image plate system using CuKα radiation from an Osmic confocal optics system. Data were processed and reduced using the program package CrystalClear provided with the instrument. Intensities were converted to structure factors using the CCP4 program TRUNCATE.Structure Determination and Refinement—Structure determination for the selenomethionine-replaced protein was accomplished using the SOLVE/RESOLVE software package (21Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 50: 760-763Google Scholar, 22Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1631) Google Scholar). Electron density was modeled as polyalanine by RESOLVE, followed by manual fitting using XTALVIEW (23McRee D.E. J. Mol. Graph. 1992; 10: 44-46Crossref Google Scholar). Refinement was performed against the structure factors from the remote wavelength using CNS (24Brunger 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 (16929) Google Scholar). Non-crystallographic restraints were not used during refinement. Refinement began with a cycle of simulated annealing (starting temperature of 3500 K) with slow cooling in increments of 25 K to a final temperature of 300 K, followed by 100 steps of conjugate gradient energy minimization. Subsequent cycles had lower initial starting temperatures (as low as 500 K). Individual thermal parameters were refined after each cycle of simulated annealing and subject to the following restraints: bonded main chain atoms, 1.5 Å2; angle main chain atoms, 2.0 Å2; bonded side chain atoms, 2.0 Å2; and angle side chain atoms, 2.5 Å2. Water molecules were automatically added using CNS if a peak greater than 3.0 σ was present in Fourier maps with coefficients (Fobs – Fcalc)eiαcalc. Refined water sites were eliminated if they were further than 3.2 Å from a hydrogen-bonding partner or if their thermal parameters exceeded 50 Å2. The contribution of the bulk solvent to structure factors was determined using the default parameters of CNS. Constants of force and geometry for the protein came from Engh and Huber (25Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2537) Google Scholar) and those for ADP from CNS resource files with appropriate modification of dihedral angles of the ribosyl moiety to maintain a 2′-endo ring conformation.For the native CAIR·ADP complex, molecular replacement was performed using AMORE with the ADP complex as the starting model. Refinement was performed as for the ADP complex. Routines in the CCP4 suite of programs were used in the calculation of surface areas and in the superposition of structures.RESULTSProtein Preparation, Data Collection, and Structure Determination—Selenium-modified and native eSS were pure on the basis of SDS-PAGE. The specific activity of the selenomethionine-substituted protein was 15 ± 1 units/mg, comparable with that of the native protein (4Nelson S.W. Binkowski D.J. Honzatko R.B. Fromm H.J. Biochemistry. 2005; 44: 766-774Crossref PubMed Scopus (17) Google Scholar). Mass spectrometry of native and selenomethionine-substituted proteins indicated 8.5 (relative to a maximum of 10) selenium atoms per monomer. SOLVE initially located 17 selenium sites, generating a phase set with a figure-of-merit of 0.37. Iterations of density modification by RESOLVE increased the figure-of-merit to 0.67. Statistics of data collection and refinement are in Tables 1 and 2.TABLE 1Statistics of data collectionInflection (E1)Peak (E2)Remote (E3)Wavelength (Å)0.979000.978840.98671Resolution (Å)46.4-2.00 (2.07-2.00)46.4-2.20 (2.28-2.00)46.4-2.00 (2.07-2.00)Reflections measured276,309215,947286,076Reflections unique40,96731,04140,775Redundancy6.74 (5.28)6.96 (7.05)7.02 (5.42)% Completeness99.7 (97.2)100.0 (100.0)99.6 (95.8)RmergeaRmerge = ΣjΣi|Iij - 〈Ij 〉|/ΣiΣjIij, where i runs over multiple observations of the same intensity, and j runs over all crystallographically unique intensities.0.133 (0.515)0.107 (0.345)0.067 (0.331)I/σ(I)7.9 (2.6)10.3 (4.5)16.3 (4.7)f′ (electrons)-15.2-9.45-4.8f″ (electrons)6.410.50.5a Rmerge = ΣjΣi|Iij - 〈Ij 〉|/ΣiΣjIij, where i runs over multiple observations of the same intensity, and j runs over all crystallographically unique intensities. Open table in a new tab TABLE 2Statistics of refinementADP complexADP·CAIR complexSpace groupP212121P212121Unit cell parametersa = 59.42, b = 67.13, c = 148.5a = 59.43, b = 67.12, c = 149.3Resolution25-2.00 (2.07-2.00)25-2.05 (2.12-2.05)No. of reflections286,076205,397No. of unique reflections40,77534,593% Completeness99.6 (95.8)90.4 (61.7)RmergeaRmerge = ΣjΣi|Iij - 〈Ij 〉| / ΣiΣjIij, where i runs over multiple observations of the same intensity, and j runs over all crystallographically unique intensities.0.067 (0.331)0.059 (0.280)No. of atoms42074095No. of solvent sites363197RfactorbRfactor = Σ||Fobs| - |Fcalc|| / Σ|Fobs|, where |Fobs| > 0.20.422.0RfreecRfree based upon 10% of the data randomly culled and not used in the refinement.24.026.3Mean B for protein (Å2)2531Mean B for ligands (Å2)2328Mean B for waters (Å2)3336Root mean square deviations Bond lengths (Å)0.0050.006 Bond angles (deg.)1.31.3 Dihedral angles (deg.)22.522.6 Improper angles (deg.)1.981.86a Rmerge = ΣjΣi|Iij - 〈Ij 〉| / ΣiΣjIij, where i runs over multiple observations of the same intensity, and j runs over all crystallographically unique intensities.b Rfactor = Σ||Fobs| - |Fcalc|| / Σ|Fobs|, where |Fobs| > 0.c Rfree based upon 10% of the data randomly culled and not used in the refinement. Open table in a new tab Overview of eSS Structure (Protein Data Bank Identifiers 2GQR and 2GQS)—An eSS homodimer occupies the crystallographic asymmetric unit. The subunits of the dimer are virtually identical with a superposition of all Cα atoms yielding a root mean square deviation of 0.34 Å for both nucleotide complexes. No electron density is present for the polyhistidyl tag. Observable electron density begins with Met1 and continues to the C terminus (Asp237). Electron density is weak only for residues 35–39 of the ADP complex but strong for the same segment in the ADP·CAIR complex.Domain 1 of the eSS fold (Fig. 1) consists of a β-sheet (strands β1-β3, β6, and β7) with its inter-strand connections (helix α1 and an anti-parallel loop β4–β5). Domain 2 consists of a β-sheet (strands β8–β13) and associated helices α2–α6. The β-sheet of domain 1 curls (like the fingers of a right hand relative to its palm) over domain 2, creating a cleft, half of which is filled by ADP-Mg2+ and the other half by CAIR. The subunits come together with 2-fold symmetry forming a dimer that buries ∼2400 Å2 of surface at the interface.The major structural difference between the ADP and ADP·CAIR complexes is the aforementioned levels of electron density associated with residues 35–39. Superposition of all Cα carbons of subunit A from the ADP and ADP·CAIR complexes gives a root mean square difference of 0.18 Å and a maximum displacement of 0.67 Å. The former value is comparable with the coordinate uncertainty of 0.25 Å determined by the CCP4 program SFCHECK. The high level of agreement occurs despite the difference in ligation, and infers selenomethionine substitution in the ADP complex causes little perturbation to the structure. The largest Cα displacements (0.7 Å) are in the loop (residues 124–130) that coordinates metal ions associated with CAIR and for residues in the vicinity of the 5′-phosphoryl group of CAIR. The conformation of the adenine nucleotide and its interactions with the protein are identical (within coordinate uncertainty) in the ADP and ADP·CAIR complexes.Comparison of eSS to tSS—eSS (237 residues) and tSS (PDB identifier 1KUT, 230 residues) share 39% sequence identity. tSS, like eSS, is a dimer (Fig. 1). Cα atoms of the eSS and tSS subunits superimpose with a root mean square deviation of ∼1.2 Å, using the sequence alignment of Fig. 2; however, the polypeptide fold associated with segment 204–221 of eSS, which includes strand β13 and helix α5, differs strikingly from that of tSS (Figs. 1 and 3). The alternative fold of tSS exposes six hydrophobic residues and increases the solvent-accessible surface area of each subunit by ∼1000 Å2 (from 1220 Å2 in eSS to 2200 Å2 in tSS).FIGURE 2Sequence alignment based on structure. Superpositions of SAICAR synthetase from yeast (ySS), T. maritima (tSS), and E. coli (eSS) determine corresponding residues. The relationship between elements of sequence and secondary structure (α-helices as cylinders and β-strands as arrows) of eSS appear immediately below its sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Variation in the folds of eSS and tSS. The superposition of subunits A and B of tSS onto eSS using the β-sheet of domain 2 reveals significant variations between subunit A (white) and subunit B (gray) of tSS, as well as an even larger conformational difference between each of the tSS subunits and subunit A of eSS (black). Subunit B of eSS (not shown) is virtually identical in conformation to subunit A. Parts of this figure were drawn with MOLSCRIPT (42Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Unlike the alternative fold of tSS, the eSS fold has an extensive network of hydrogen bonds. Interacting residues fall in two clusters: Asp202, Arg231, and Thr205 and Arg39, Asp175, Arg199, Asp210, Lys211, Asp212, Arg213, and Arg215. The latter more extensive cluster apparently anchors helix α5 with respect to domains 1 and 2, while positioning hydrophilic side chains in the active site cleft of eSS. In contrast, helix α5 in tSS is displaced relative to that of eSS (Fig. 3), taking residues corresponding to Arg199, Lys211, and Arg215 away from the active site. Most of the residues in segment 204–221 of eSS are conserved among microbial SAICAR synthetases; for instance, Asp175, Arg199, Asp210, Lys211, Asp212, and Arg215 are conserved and present in tSS.Comparison of eSS to ySS—SAICAR synthetase from S. cerevisae (ySS) has 69 more amino acids than eSS, appearing primarily as insertions before residues 1, 77, 105, and 221 of the E. coli synthetase (Figs. 1 and 2). Neglecting insertions, eSS and ySS are 27% identical in sequence, and superimpose with a root mean square deviation of 3.3 Å. The first and second sequence insertions come together in ySS (PDB identifiers 1OBD, 1OBG, and 1A48), where they define a putative binding site for AMP. (AMP appears in good electron density only at a lattice contact in 1OBG. Hence, the functional significance of the first two insertions in the ySS sequence remains unclear.) The third insertion occurs at the subunit interface of the eSS dimer and probably blocks the dimerization of ySS subunits. The fourth insertion extends the helix corresponding to α5 of eSS and the connecting segments at the N- and C-terminal ends of that helix. The fourth segment replaces residues 204–221 in eSS but, nonetheless, retains a functional active site.Adenine Nucleotide Interactions—ADP-Mg2+ binds to eSS in an anti-conformation (Fig. 4). Val15, Leu24, Leu26, and Val81 are in contact with one side of the adenine base, whereas Met86 packs against the other. Atom N-1 of ADP binds to the backbone amide group of Leu84, and atom N-6 binds to the backbone carbonyl group of Lys82 and the side chain of Gln69 (Table 3). No side chain interaction between atom N-6 and the protein was reported for ySS (PDB identifiers 1OBG and 1OBD); however, His72 of the ySS structurally corresponds to Gln69 of eSS and is in a position to interact with the adenine nucleotide. This position is conserved as glutamine or histidine in microbial systems.FIGURE 4Enzyme-bound ADP. Left and center, stereoview of ADP in which the dotted lines represent donor-acceptor interactions. The filled circle represents Mg2+, and open circles are water molecules coordinated to the metal. Parts of this figure were drawn with MOLSCRIPT (42Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar). Right, omit electron density covering the hydrated ADP-Mg2+ molecule bound at the active site of eSS. The contour level is at 1 σ with a cutoff radius of 1 Å. Mg2+ is the filled circle and water molecules are crosses. Dotted lines indicate coordinate bonds to the metal. Parts of this figure were drawn with XTALVIEW (23McRee D.E. J. Mol. Graph. 1992; 10: 44-46Crossref Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 3Selected polar contacts involving ligandsLigand atomContact partnerDistanceÅADP N-1Leu84 N3.06 N-6Gln69 OE12.62Lys82 O2.98 N-7Asp191 N3.03 O-2′Glu179 OE22.92 O-1ALys11 N3.03Ala12 N2.70Lys13 N2.73 O-2ALys13 NZ2.78 O-1BLys11 N2.75Lys11 NZ2.88 O-2BLys123 NZ2.91CAIR O-3ASer100 OG2.64Arg94 NH22.61 O-2AArg94 NH13.15Ser100 N2.93Arg199 NH12.96 O-3′Asp175 OD12.62 O-2′Arg215 NH22.77 Open table in a new tab The ribosyl moiety is C2′-endo, as observed for the adenine nucleotides in ySS structures. Atom O-2′ of the ribose binds to Glu179, corresponding to an equivalent interaction with Glu219 in ySS.The polyphosphoryl group of the adenine nucleotide interacts with strands β1 and β2, which together constitute a P-loop motif (26Dever T.E. Glynias M.J. Merrick W.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1814-1818Crossref PubMed Scopus (463) Google Scholar, 27Saraste M. Sibbald P.R. Wittinghofer A. Trends Biochem. Sci. 1990; 15: 430-434Abstract Full Text PDF PubMed Scopus (1733) Google Scholar). The α-phosphoryl group interacts with backbone amide groups of Lys11, Ala12, and Lys13, with atom NZ of Lys13, and with Mg2+ (hereafter, Mg2+ site 1). The β-phosphoryl group interacts with the backbone amide group and side chain of Lys11, the amino group of Lys123, and Mg2+ site 1. Four water molecules complete the octahedral coordination sphere of the Mg2+ site 1 (Table 4). Lys13, Glu179, Lys177, and Asp191 form additional hydrogen bonds with the hydrated magnesium.TABLE 4Coordination distances and coordinating atoms of Mg2+ at sites 1–3Mg2+ site 1Mg2+ site 2Mg2+ site 3ADP O-2A2.21Asp129 OD22.12As" @default.
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• W2106722820 title "Nucleotide Complexes of Escherichia coli Phosphoribosylaminoimidazole Succinocarboxamide Synthetase" @default.
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