Crystal structure of archaeal highly thermostable L-aspartate dehydrogenase/NAD/citrate ternary complex Kazunari Yoneda1, Haruhiko Sakuraba2, Hideaki Tsuge3, Nobuhiko Katunuma3 and Toshihisa Ohshima1 Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Japan Institute for Health Sciences, Tokushima Bunri University, Japan Keywords aromatic pair interaction; hyperthermostable L-aspartate dehydrogenase; ion-pair interaction; NAD biosynthesis Correspondence T Ohshima, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan Fax: +81 92 642 3059 Tel: +81 92 642 3053 E-mail: ohshima@agr.kyushu-u.ac.jp (Received 13 May 2007, revised 26 June 2007, accepted 28 June 2007) doi:10.1111/j.1742-4658.2007.05961.x The crystal structure of the highly thermostable l-aspartate dehydrogenase (l-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate The dimeric structure of A fulgidus l-aspDH was refined at ˚ a resolution of 1.9 A with a crystallographic R-factor of 21.7% (Rfree ¼ 22.6%) The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site Structural comparison of the A fulgidus l-aspDH ⁄ NAD ⁄ citrate ternary complex and the Thermotoga maritima l-aspDH ⁄ NAD binary complex showed that A fulgidus l-aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding Like T maritima l-aspDH, the A fulgidus enzyme is highly thermostable But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A fulgidus l-aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T maritima enzyme Thus stabilization of these two l-aspDHs appears to be achieved in different ways This is the first detailed description of substrate and coenzyme binding to l-aspDH and of the molecular basis of the high thermostability of a hyperthermophilic l-aspDH In prokaryotes, de novo NAD biosynthesis generally proceeds via a condensation reaction between l-aspartate and dihydroxyacetone phosphate that is catalyzed by two enzymes: l-aspartate oxidase (LAO; the nadB gene product) and quinolinate synthase (QS; the nadA gene product) [1] LAO catalyzes the oxidation of l-aspartate to iminoaspartate, after which QS catalyzes the condensation of iminoaspartate with dihydroxyacetone phosphate to produce quinolinate Quinolinate is then converted to nicotinate mononucleotide by quinolinate phosphoribosyltransferase (the nadC gene product), which is followed by conversion to NAD via a metabolic sequence involving two enzymes: nicotinate mononucleotide adenylyltransferase and NAD synthase [2] We recently detected the presence of LAO in Pyrococcus horikoshii OT-3, an anaerobic hyperthermophilic archaeon that grows optimally at 98 °C [3] In addition to the oxidase reaction, this LAO also catalyzed FAD-dependent l-aspartate dehydrogenation using fumarate as an electron acceptor This was the first example of an LAO produced in either the archaeal domain or an obligate anaerobic organism Thereafter, we identified genes encoding homologs of four other enzymes involved in de novo NAD biosynthesis in the P horikoshii genome and found that nadB forms Abbreviations LAO, L-aspartate oxidase; L-aspDH, L-aspartate dehydrogenase; MIRAS, multiple isomorphous replacement with anomalous scattering; QS, quinolinate synthase FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4315 Crystal structure of L -aspDH from A fulgidus K Yoneda et al an operon with nadA, nadC and three other unknown genes We therefore proposed that a de novo NAD biosynthetic pathway functions in P horikoshii under anaerobic conditions [3] More recently, a previously unknown amino acid dehydrogenase, l-aspartate dehydrogenase (l-aspDH; the TM1643 gene product), was identified in a hyperthermophilic bacterium Thermotoga maritima by Yang et al based on the 3D structure of the gene product [4] This enzyme catalyzes NAD(P)-dependent dehydrogenation of l-aspartate to produce iminoaspartate Because the gene encoding l-aspDH also forms an operon with nadA and nadC within the T maritima genome, those authors suggested that l-aspDH catalyzes the first step in the de novo biosynthesis of NAD in this organism Thus, two different types of oxidoreductase may have evolved to catalyze the first step of de novo NAD biosynthesis in prokaryotes [4] Bearing in mind that hyperthermophilic archaea are phylogenetically ancient, our interest in the phylogenetic relationship between LAO and l-aspDH led us to screen for an l-aspDH homolog in the genomes of hyperthermophilic archaea Within the genomic sequence of an anaerobic hyperthermophilic archaeon, Archaeoglobus fulgidus, we found a gene (AF1838) whose predicted amino acid sequence exhibits 38% identity with that of T maritima l-aspDH In addition, we showed that the gene product expressed in Escherichia coli acts as a highly thermostable l-aspDH [5] Yang et al determined the structure of T maritima l-aspDH in the presence of NAD and analyzed the structural features responsible for its dehydrogenase activity [4] T maritima l-aspDH does not share structural similarity with the superfamilies that include the l-leucine, l-valine, l-glutamate and l-phenylalanine dehydrogenase, and l-alanine dehydrogenase, despite all these enzymes catalyzing similar chemical reactions In addition, the structure of T maritima l-aspDH has only low similarity to aspartate semialdehyde dehydrogenase, inositol 1-phosphate synthase and dihydrodipicolinate dehydrogenase This enzyme thus appears to represent a new class of amino acid dehydrogenase However, details about the molecular strategy underlying its high thermal stability, as well as the manner in which substrate and coenzyme are bound by the enzyme, are still unclear Therefore, our aim was to determine the structure of the A fulgidus l-aspDH in complex with NAD and a substrate analog, citrate Factors that could stabilize the enzyme were then compared with those in the T maritima l-aspDH, and the structural features that appear to be responsible for the high thermostability of each enzyme are discussed Finally, we describe a substrate-induced conforma4316 tional change in the ternary complex of A fulgidus l-aspDH Results and Discussion Overall structure The structure of A fulgidus l-aspDH was determined using multiple isomorphous replacement with anomalous scattering (MIRAS) and was refined at a resolu˚ tion of 1.9 A (Table 1) The asymmetric unit consisted of one homodimer with a solvent content of 39.3%, which corresponds to a Matthew’s coefficient [6] of ˚ 2.0 A3ỈDa)1 The model contained 236 ordered amino acid residues in each subunit and 136 water molecules ˚ The two almost identical (r.m.s.d ¼ 0.42 A) subunits ˚ had approximate dimensions of 35 · 53 · 44 A and were related by a twofold noncrystallographic rotation axis and associated closely through antiparallel b sheets (b11 and b11¢) (Fig 1A) Each monomer consisted of two domains: an N-terminal coenzyme-binding domain (a1–a5, a10, b1–b6, b12–b14), which formed a classical Rossmann-fold motif, and a C-terminal domain (a6– a9, b7–b11), in which both the catalytic activity and formation of the homodimer were mediated (Fig 1B) Overall domain organization was similar to that of T maritima l-aspDH, but with several critical differences in the structural details Structural comparison of A fulgidus and T maritima L-aspDHs When we compared the structures of the A fulgidus l-aspDH ⁄ NAD ⁄ citrate ternary complex and the T maritima l-aspDH ⁄ NAD binary complex, we found that the overall folds of A fulgidus and T maritima l-aspDH were similar (Fig 3A), which was expected, given their relatively high sequence identity (38%) (Fig 2) The internal structure of the A fulgidus enzyme was basically the same as that of the T mari˚ tima enzyme (r.m.s.d ¼ 1.9 A for the Ca atoms of 223 residues), although a marked difference was observed in the position of three loops in the catalytic domain When the Rossmann-fold domain of the A fulgidus l-aspDH structure was superimposed on that of the T maritima l-aspDH structure, we observed a large shift in the positions of two loops (loop 1: R133– G143, loop 2: V180–I188 in the A fulgidus l-aspDH) toward the active site cavity (Fig 3) The average movements of loops and were estimated to be 3.2 ˚ and 3.6 A, and the largest movements of loops and ˚ ˚ were estimated to be 6.7 A (K142) and 5.8 A (E183), respectively As described below, this large FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS K Yoneda et al Crystal structure of L -aspDH from A fulgidus ˚ Table Statistics on data collection, phase determination and refinement The crystal belongs to space group P21212 with a ¼ 47.52 A, ˚ ˚ b ¼ 89.58 A, c ¼ 100.49 A Native Data collection ˚ Maximum resolution (A) Total reflections Unique reflections Redundancy Completenessd (%) Rsymd,e (%) < I ⁄ r (I) > d MIRAS phasing FOM Refinement ˚ Resolution range (A) f Rcryst (%) Rfreeg (%) No of protein atoms No of water molecules No of NAD No of citrate ˚ RMSD bond lengths (A) RMSD bond angles (°) ˚ Average B-factors (A2) Protein NAD Citrate Water Hg1a Hg2b Hg3c 1.9 237826 34 001 7.0 98.0 (87.6) 5.5 (23.9) 15.7 (5.9) 2.0 212964 55997 3.8 99.9 (99.1) 6.4 (22.9) 13.0 (4.4) 2.0 202467 54558 3.7 97.2 (85.6) 5.1 (21.3) 13.7 (4.0) 1.79 289592 77004 3.8 98.2 (86.7) 5.4 (25.4) 15.7 (3.4) 0.68 (32.6–2.0) 32.6–1.90 21.7 22.6 3686 136 2 0.011 1.5 29.5 26.9 34.2 33.4 a Hg1, ethyl mercuric phosphate b Hg2, 1,4-diacetoxymercuri-2,3-dimethoxybutane c Hg3, phenylmercury acetate d Values in parentheses are for the last resolution shell e Rsym ¼ Sh Si | I i (h) – < I (h) > | ⁄ Sh Si | I i (h) |,where Ii (h) is the intensity measurement for a reflection h and < I (h) > is the mean intensity for this reflection f Rcryst ¼ ShjjFobsj–jFcalcjj ⁄ ShjFobsj g The Rfree was calculated with randomly selected reflections (10%) conformational change may be essential for substrate binding In addition, loop (P208–S216), which is disordered in the T maritima enzyme, was clearly observable in the structure of A fulgidus enzyme and formed a flap over the active site cavity (Fig 3) It has been proposed that the structure of T maritima l-aspDH assumes an open configuration [4] Our results strongly suggest that loop functions as the substratebinding site and the structure of A fulgidus l-aspDH assumes a closed configuration Substrate binding In the electron-density map of A fulgidus l-aspDH obtained from our preliminary experimental data, we noticed an extra density within the active site cavity and found that a citrate molecule could be modeled into that density after construction and refinement of the peptide chain The map clearly defined the precise orientation of the citrate (Fig 4A): the two oxygen atoms in the C-1 carboxyl group of the citrate form hydrogen bonds with the side chain of K134, a proton at the main chain amide of L161, and a water molecule (WAT33); two oxygen atoms in the C-5 carboxyl group are located within hydrogen-bonding distance of the side chain of N162 and backbone amide protons in N162 and V163; oxygen atoms in the C-6 carboxyl group form hydrogen and ionic bonds with the side chains of N187, H189 and S216, and the main chain amide proton in S216; and an oxygen atom in the C-3 hydroxyl group forms a hydrogen bond with the side chain of K134 Together, these bonds tightly hold the citrate near the nicotinamide ring of NAD Based on the structure of citrate, we modeled the l-aspartate molecule into the active site of A fulgidus l-aspDH (Fig 4B) and then minimized the energy of the complex using insight ii (Biosym ⁄ MSI, San Diego, CA) Within this structure, two oxygen atoms of the Ca carboxylate are situated within hydrogen- and ionic-bonding distance of the side chains of N187, H189 and S216, and the main chain amide proton of S216 In addition, the oxygen atoms of the Cb carboxyl group FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4317 Crystal structure of L -aspDH from A fulgidus K Yoneda et al A B Fig Structure-based amino acid sequence alignment of A fulgidus and T maritima L-aspDHs (AF, Archaeoglobus fulgidus; TM, Thermotoga maritima) Sequences were aligned using CLUSTAL W [35] Boxes represent conserved residues in the enzymes The residues that are disordered in T maritima L-aspDH are shown in purple The residues involved in citrate and NAD binding are shown in blue and red, respectively The secondary structural assignments for the A fulgidus L-aspDH structure are shown above the alignment in green; those for the T maritima L-aspDH structure are shown below the alignment in blue Fig (A) The A fulgidus L-aspDH dimeric model viewed down the twofold noncrystallographic rotation axis The A and B subunits are shown in rainbow and gray, respectively (B) Ribbon plot of the A fulgidus L-aspDH monomer The NAD binding and catalytic domains are shown in green and blue, respectively NAD (magenta) and citrate (yellow) are shown as stick models are within hydrogen-bonding distance of the side chain of K134, the main chain amide proton of L161, and a water molecule (WAT33) All of the residues are conserved in T maritima l-aspDH, except for L161, which is replaced with Ile Within the active site of the T maritima enzyme, however, we found that the side chains of residues corresponding to K134 in loop 1, N187 in loop and S216 in loop are far removed from the l-aspartate molecule, and it does not appear that hydrogen bonds are formed with the carboxyl groups of the substrate (Figs 3A and 4B) This 4318 suggests that the large movement of loops and 2, in addition to the formation of a flap by loop 3, may induce the suitable positioning of the three residues (K134, N187 and S216) for substrate binding and the expression of l-aspDH’s catalytic activity In general, NAD(P)H-dependent dehydrogenases show pro-R or pro-S stereospecificity for hydrogen removal from the C4 position of the nicotinamide moiety of NAD(P)H In our binding model, the re-face of the nicotinamide ring is in front of the a-hydrogen atom of substrate (Fig 4B), and that is in good agreement with our earlier finding that A fulgidus l-aspDH belongs to the dehydrogenase group with pro-R-specific hydrogen transfer [5] Cofactor binding The electron density corresponding to the NAD coenzyme bound within the active site was very clear, which enabled us to place the ligand with reasonable accuracy (Fig S1) The map enabled clear positioning of the adenine ring, identification of the C2¢-endo FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS K Yoneda et al A B Crystal structure of L -aspDH from A fulgidus conformation of the two ribose rings, clear definition of the backbone phosphate groups and assignment of an anti conformation to the glycosidic bond linking the nicotinamide ring and its associated ribose moiety N7, N1 and N6 of the adenine base, respectively, formed hydrogen bonds with the side chains of S59 and Y66 and, via a water molecule (WAT26), with the side chain of D65 The hydroxyl groups of the adenine ribose interact with R33 using side-on geometry A glycine-rich motif, GXGXXG, which lies close to the adenine ribose, dictates the nature of the hydrogen bonding between the main chain and the adenine ribose moiety [7] In A fulgidus l-aspDH, this motif is recognized at the position of the 7–12th amino acids from the N-terminus (Fig 2) It is known that the occurrence of an aspartic or glutamic acid residue at the C- or N-terminus of the second b strand of the bab fold is a common feature of NAD(P)-dependent dehydrogenases [8–12] The acidic residue plays an important role in the formation of hydrogen bonds with the adenine ribose hydroxyl groups of the cofactor [13,14] In A fulgidus l-aspDH, D31, which is at the N-terminal end of b2, forms hydrogen bonds with the 2¢ and 3¢ hydroxyl groups of the adenine ribose of NAD (Fig 5) The adenine phosphate interacts with A10, I11, a water molecule (WAT119) and, via water molecules, with R33 (WAT125) and A57 (WAT5) In addition, the nicotinamide phosphate interacts with N212, T215 and a water molecule (WAT119) and, via a water molecule (WAT5), with A57 The hydroxyl groups of the nicotinamide ribose interact with the side chain of S81, N162 and backbone oxygen of A58 The hydrogen-bonding pattern between the enzyme and NAD is completed by interactions between the carboxyamide moiety of the nicotinamide ring and the main-chain NH of A111, the main-chain oxygen of L80, the side chain of T166 (via water molecule WAT38), and the main-chain oxygen of A108 These hydrogen bonds lead to an anti conformer for the glycosidic bond between the nicotinamide ring and its associated ribose, and enable the 4-pro-R hydrogen atom to be involved in the hydride-transfer step Accessible surface area and hydrogen bonds Fig Comparison of the structures of A fulgidus L-aspDH and T maritima L-aspDHs (A) The superimposed Ca-traces of A fulgidus L-aspDH and T maritima L-aspDH; the structures of the two enzymes are shown in green and magenta, respectively NAD (magenta) and citrate (yellow) molecules are shown as sphere models (B) Scheme around the substrate binding loop in the catalytic domain The citrate molecule is shown as a stick model in yellow Oxygen and nitrogen atoms are shown in red and blue, respectively L-AspDHs are colored as in (A) It is thought that, in general, a reduction in the solvent-accessible surface area and an increase in the fraction of buried hydrophobic atoms are the stabilizing principles that serve as the basis for protein thermostability [15] As shown in Table 2, the total solventaccessible surface area of A fulgidus l-aspDH ˚ ˚ (monomer, 12 665 A2; dimer, 20 685 A2) is similar to ˚ that of T maritima l-aspDH (monomer, 12 892 A2; FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4319 Crystal structure of L -aspDH from A fulgidus K Yoneda et al A Fig Stereoview of the citrate coordinated within the active site and the proposed binding model for the L-aspartate molecule (A) Schematic representation depicting the interactions between citrate and the protein The networks of hydrogen bonds are shown by dotted lines The citrate molecule is shown as a stick model in yellow The final rA-weighted 2|Fo| ) |Fc| electron-density map for citrate is shown at the 1r level (B) Comparison of the A fulgidus L-aspDH active site pocket with that of T maritima L-aspDH and the proposed binding model of the L-aspartate The C4 atom of the pyridine ring (a hydride acceptor site), and si- and re-faces are labeled The L-aspartate molecule is shown as a stick model in cyan The structures of A fulgidus and T maritima L-aspDH are shown in green and white, respectively Atoms are colored as described for Fig B Fig Stereo representation of NAD bound to A fulgidus L-aspDH Residues that interact with NAD are labeled The networks of hydrogen bonds are shown as dotted lines Atoms are colored as described for Fig ˚ dimer, 21 381 A2) Likewise, the total interface area ˚ between A fulgidus l-aspDH subunits A–B (2322 A2) is similar to that between the T maritima l-aspDH ˚ subunits (2201 A2), and the total hydrophobic areas ˚ of the interfaces are about the same (972–973 A2) 4320 (Table 2) There was also no significant difference in the number of hydrophobic residues (108 and 113 residues in A fulgidus and T maritima l-aspDHs, respectively) between the two enzymes Using insight ii (Biosym ⁄ MSI), the number of hydrogen bonds within FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS K Yoneda et al Crystal structure of Table Comparison of ion-pair interactions, aromatic pair interactions, accessible surface areas, and hydrogen bonding in A fulgidus and T maritima L-aspDHs A fulgidus PDB code ˚ Resolution (A) No intrasubunit ion pairs per subunit No intersubunit ion pairs Ion pair network No intrasubunit aromatic pairs per subunit No intersubunit aromatic pairs Largest aromatic pair network ˚ Solvent-accessible surface area (A2) Monomer Dimer Interface Hydrophobic interface No intrasubunit H bond per subunit No intersubunit H bond T maritima 2DC1 1.9 16 1J5P 1.9 1·4 residues not observed 2·4 residues · residues 12665 20685 2322 973 149 12892 21381 2201 972 131 26 28 11 the A fulgidus and T maritima l-aspDH monomers were determined to be 149 and 131, respectively, and the number of intersubunit hydrogen bonds was 26 and 28, respectively Again, the two enzymes showed considerable similarity L -aspDH from A fulgidus to the thermostability of these enzymes [19–21] We examined the structural characteristics of the A fulgidus [5] and T maritima l-aspDHs from the aspect of high thermostability The stability of T maritima l-aspDH has not been reported to date Thus, we expressed the enzyme gene in E coli and purified the recombinant enzyme to homogeneity Thermostability of the enzyme was then compared with that of A fulgidus l-aspDH The half-life of A fulgidus l-aspDH (t1 ⁄ ¼ 10.0 min) was similar to that of T maritima enzyme (t1 ⁄ ¼ 10.7 min) at 100 °C, indicating that the thermostabilities of the two enzymes are comparable ˚ Using a cut-off distance of 4.0 A between oppositely charged residues, we calculated that A fulgidus l-aspDH contained 16 intrasubunit ion pairs, whereas T maritima l-aspDH contained only nine (Table 2) In addition, three major intersubunit ion-pair interactions were observed in A fulgidus l-aspDH: E201– R203¢, R203–E201¢ and R95–E232¢ (the prime indicates the neighboring subunit in the dimer) An ion pair between E232 and R95¢ could not be observed because of the poor electron density of the side chains Four (E201, R203, E201¢, and R203¢) of the residues are located within b11 and b11¢, and form a four-residue ion-pair network between the A and B subunits (Fig 6) In the T maritima l-aspDH, the charged R95 is replaced by aromatic F93, which is involved in the largest aromatic pair network in T maritima enzyme (see below) In addition, T maritima l-aspDH contains no ion-pair networks (Table 2) Comparison of the amino acid compositions The higher numbers of Pro at the N1 position or Ala and lower numbers of b-branched residues (Val, Thr and Ile) are known to correlate significantly with the thermostability of proteins by stabilizing their a helices [16–18] The total numbers of Pro at the N1 position and b-branched residues were and 15, respectively, in the a helices of A fulgidus l-aspDH, and and 18, respectively, in the T maritima enzyme By contrast, the total numbers of Ala residues in the a helices of A fulgidus and T maritima l-aspDHs were 15 and 5, respectively Thus, A fulgidus and T maritima l-aspDHs not differ significantly with respect to their Pro or b-branched residue contents; however, the relatively high Ala content in the a helices of the A fulgidus l-aspDH might contribute to its thermostability Aromatic pair interaction Aromatic interactions are also known to participate in stabilizing protein structure [22,23] A pair of aromatic Ion-pair interaction Recent studies of the structures of hyperthermophilic proteins have shown that the number of ion pairs and the formation of ion networks contribute significantly Fig The intersubunit ion pair network in A fulgidus L-aspDH Residues belonging to the A and B subunits are shown in green and magenta, respectively Atoms are colored as described for Fig FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4321 Crystal structure of L -aspDH from A fulgidus K Yoneda et al interactions contributes between )0.6 and )1.3 kcalỈ mol)1 to protein stability [24] Using a cut-off distance ˚ between the aromatic ring centers of 7.0 A, we determined that there are six intrasubunit aromatic pairs in A fulgidus l-aspDH (Table 2) By contrast, there are 11 aromatic pairs in T maritima l-aspDH The most extensive aromatic pair network (seven residues; Y75, F83, F88, F92, F93, F105, and F240¢) is located in the Rossmann-fold domain (Fig S2), and two of those residues (F105 and F240¢) form an intersubunit aromatic pair By contrast, the largest aromatic pair network identified in A fulgidus l-aspDH is composed of only four residues (F14, W18, F24, and Y217) and is located on the surface of the Rossmann-fold domain (Fig S2) It is noteworthy that whereas five major intersubunit aromatic pair interactions (F105–F240¢, F122–Y225¢, F206–F206¢, Y225–F122¢, and F240– F105¢) were observed in T maritima l-aspDH, there are no intersubunit aromatic pairs in the A fulgidus enzyme (Fig S2) Structure-based sequence alignment showed that with the exception of F83 and F88, the residues forming the largest aromatic pair network in T maritima l-aspDH were replaced by nonaromatic residues in A fulgidus l-aspDH (Fig 7) Thus, while it appears that intersubunit ion-pair interactions are the primary mediators of the high thermostability of A fulgidus l-aspDH, the relatively large number of intersubunit aromatic pairs suggests they are the major mediators of T maritima l-aspDH thermostability Experimental procedures Protein expression and purification Expression of the gene encoding A fulgidus l-aspDH in E coli and its purification from the recombinant cells was carried out as described previously [5] B Fig The largest aromatic pair network, formed by seven residues in T maritima L-aspDH (A), and the equivalent positions in A fulgidus L-aspDH (B) Residues belonging to the A and B subunits are shown in green and magenta, respectively Atoms are colored as described for Fig Na2HPO4, 39.5 mm citric acid), 5% (v ⁄ v) polyethylene glycol 3000 (PEG 3000), 10% (v ⁄ v) glycerol and 22% (v ⁄ v) 1.2-propanediol Crystallization For the crystallization trails, purified A fulgidus l-aspDH was dialyzed against 10 mm potassium phosphate buffer (pH 7.2) containing 0.2 m NaCl Crystallization of l-aspDH was accomplished using the sitting-drop vapordiffusion method, and the initial screening was carried out using Crystal Screen Cryo (Hampton Research, Aliso Viejo, CA, USA) at 20 °C The crystal obtained belonged to the orthorhombic space group P21212, and the unit cell param˚ ˚ ˚ eters were a ¼ 47.52 A, b ¼ 89.58 A, c ¼ 100.49 A and a ¼ b ¼ c ¼ 90° The crystals were grown in sitting drops in which lL of enzyme solution (14.5 mgỈmL)1) containing mm NAD was mixed with lL of mother liquor containing 100 mm phosphate-citrate buffer pH 4.2 (60.5 mm 4322 A Data collection Crystals were coated with a layer of viscous oil (Paratone-N) and transferred into a stream of nitrogen gas for data collection at 100 K Diffraction data were collected at a reso˚ lution of 1.9 A on beamline KEK-NW12 at the Photon Factory (Tsukuba, Japan) using monochromatized radia˚ tion at k ¼ 1.0 A and an ADSC Quantum 210 CCD detector (Area Detector Systems, San Diego, CA, USA) The oscillation angle per image was set to 1°, and the data were processed using HKL 2000 [25] Heavy atom derivatives were prepared by soaking the crystals for 24 h in mother FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS K Yoneda et al liquor containing 0.5 mm ethyl mercuric phosphate, mm 1,4-diacetoxymercuri-2,3-dimethoxybutane or mm phenylmercury acetate Crystal structure of L -aspDH from A fulgidus determined at appropriate intervals using a standard assay as described previously [5] Acknowledgements Phasing and refinement The native, ethyl mercuric phosphate, 1,4-diacetoxymercuri2,3-dimethoxybutane, and phenylmercury acetate data sets were used for phase calculation (Table 1), which was accomplished by MIRAS using solve [26] The MIRAS ˚ map at 1.9 A was subjected to maximum-likelihood density modification, followed by autotracing using resolve [27] An initial model was built using xtal view [28], after which several cycles of rigid-body refinement, positional refine˚ ment and simulated annealing were performed at 1.9 A resolution using refmac [29] and cns [30] The model was adjusted in xtal view using both |Fo| ) |Fc| and 2|Fo| ) |Fc| maps NAD and citrate molecules were clearly visible in both the rA-weighted |Fo| ) |Fc| and 2|Fo| ) |Fc| maps, and these molecules were included in the latter part of the refinement The current model contains 472 residues (A 1–236 and B 1–236), 136 water molecules, two NAD and two citrate molecules The model geometry was analyzed using procheck [31], and 91.2% of the nonglycine residues were in the most favored region of the Ramachandran plot and 8.8% in the additionally allowed region Molecular graphics were created using pymol (http://pymol.sourceforge.net/) The coordinates and structure factors of A fulgidus l-aspDH complexed with NAD and citrate have been deposited in the Protein Data Bank with the accession code 2DC1 Structural analysis and comparison Ion-pair interactions within the two structures were identified using the WHAT IF web server [32] with a cut-off ˚ distance ¼ 4.0 A [33] between oppositely charged residues Aromatic interactions were defined using a cut-off dis˚ tance of 7.0 A between the aromatic ring centers [22] Hydrogen bonds were identified using the program insight ii (Biosym ⁄ MSI, San Diego, CA, USA) Hydrogen was added to the coordinates of the proteins and the calculated hydrogen bonds (the distance between a calculated hydrogen position and model oxygen or nitrogen ˚ atom was < 3.0 A, and the angle between the proton donor and acceptor is > 120°) were measured using the criterion of insight ii The solvent accessible surface area was calculated using the program grasp [34] Thermal stability To assess the thermostability, the A fulgidus or T maritima l-aspDH (0.1 mgỈmL)1) in 10 mm potassium phosphate buffer (pH 7.2) containing 0.2 m NaCl was incubated at 100 °C, after which the residual activity of the enzymes was Data collection was performed at the Photon Factory (Tsukuba, Japan) We thank Drs K Demura, N Matsugaki, N Igarashi and S Wakatsuki for their kind assistance with the data collection This work was supported in part by the ‘National Project on Protein Structural and Functional Analysis’ promoted by the Ministry of Education, Science, Sports, Culture, and Technology of Japan and by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science References Nasu S, Wicks FD & Gholson RK (1982) The mammalian enzyme which replaces B protein of E coli quinolinate synthetase is d-aspartate oxidase Biochim Biophys Acta 704, 240–252 Rizzi M & Schindelin H (2002) Structural biology of enzymes involved in NAD and molybdenum cofactor biosynthesis Curr Opin Struct Biol 12, 709–720 Sakuraba H, Satomura T, Kawakami R, Yamamoto S, Kawarabayasi Y, Kikuchi H & Ohshima T (2002) l-Aspartate oxidase is present in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3: characteristics and role in the de novo biosynthesis of nicotinamide adenine dinucleotide proposed by genome sequencing Extremophiles 6, 275–281 Yang Z, Savchenko A, Yakunin A, Zhang R, Edwards A, Arrowsmith C & Tong L (2003) Aspartate dehydrogenase, a novel enzyme identified from structural and functional studies of TM1643 J Biol Chem 278, 8804–8808 Yoneda K, Kawakami R, Tagashira Y, Sakuraba H, Goda S & Ohshima T (2006) The first archaeal l-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: gene cloning and enzymological characterization Biochim Biophys Acta 1764, 1087–1093 Matthews BW (1968) Solvent content of protein crystals J Mol Biol 33, 491–497 Baker PJ, Britton KL, Rice DW, Rob A & Stillman TJ (1992) Structural consequences of sequence patterns in the fingerprint region of the nucleotide binding fold Implications for nucleotide specificity J Mol Biol 228, 662–671 Scrutton NS, Berry A & Perham RN (1990) Redesign of the coenzyme specificity of a dehydrogenase by protein engineering Nature 343, 38–43 Feeney R, Clarke AR & Holbrook JJ (1990) A single amino acid substitution in lactate dehydrogenase improves the catalytic efficiency with an FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4323 Crystal structure of 10 11 12 13 14 15 16 17 18 19 20 21 22 23 L -aspDH from A fulgidus K Yoneda et al alternative coenzyme Biochem Biophys Res Commun 166, 667–672 Chen Z, Lee WR & Chang SH (1991) Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase Eur J Biochem 202, 263–267 Fan F, Lorenzen JA & Plapp BV (1991) An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme Biochemistry 30, 6397– 6401 Nishiyama M, Birktoft JJ & Beppu T (1993) Alteration of coenzyme specificity of malate dehydrogenase from Thermus flavus by site-directed mutagenesis J Biol Chem 268, 4656–4660 Baker PJ, Sawa Y, Shibata H, Sedelnikova SE & Rice DW (1998) Analysis of the structure and substrate binding of Phormidium lapideum alanine dehydrogenase Nat Struct Biol 5, 561–567 Wierenga RK, de Maeyer MCH & Hol WGJ (1985) Interaction of pyrophosphate moieties with a-helixes in dinucleotide-binding proteins Biochemistry 24, 1346– 1357 Chan MK, Mukund S, Kletzin A, Adams MW & Rees DC (1995) Structure of a hyperthermophilic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase Science 267, 1463–1469 Yun RH, Anderson A & Hermans J (1991) Proline in a-helix: stability and conformation studied by dynamics simulation Proteins 10, 219–228 Padmanabhan S, Marqusee S, Ridgeway T, Laue TM & Baldwin RL (1990) Relative helix-forming tendencies of nonpolar amino acids Nature 344, 268–270 Facchiano AM, Colonna G & Ragone R (1998) Helix-stabilizing factors and stabilization of thermophilic proteins: an X-ray based study Protein Eng 11, 753–760 Hennig M, Darimont B, Sterner R, Kirschner K & ˚ Jansonius JN (1995) 2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability Structure 3, 1295–1306 DeDecker BS, O’Brien R, Fleming PJ, Geiger JH, Jackson SP & Sigler PB (1996) The crystal structure of a hyperthermophilic archaeal TATA-box binding protein J Mol Biol 264, 1072–1084 Lim JH, Yu YG, Han YS, Cho S, Ahn BY, Kim SH & Cho Y (1997) The crystal structure of an Fe-superoxide dismutase from the hyperthermophile Aquifex pyrophilus ˚ at 1.9 A resolution: structural basis for thermostability J Mol Biol 270, 259–274 Burley SK & Petsko GA (1985) Aromatic–aromatic interaction: a mechanism of protein structure stabilization Science 229, 23–28 Kannan N & Vishveshwara S (2000) Aromatic clusters: a determinant of thermal stability of thermophilic proteins Protein Eng 13, 753–761 4324 24 Serrano L, Bycroft M & Fersht AR (1991) Aromatic– aromatic interactions and protein stability Investigation by double-mutant cycles J Mol Biol 218, 465–475 25 Otwinowski Z & Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode Methods Enzymol 276, 307–326 26 Terwilliger TC & Berendzen J (1999) Automated MAD and MIR structure solution Acta Crystallogr D55, 849–861 27 Terwilliger TC (2000) Maximum-likelihood density modification Acta Crystallogr D56, 965–972 28 McRee DE (1999) XtalView ⁄ Xfit – A versatile program for manipulating atomic coordinates and electron density J Struct Biol 125, 156–165 29 Murshudov GN, Vagin AA & Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr D53, 240–255 30 Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS et al (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination Acta Crystallogr D54, 905–921 31 Laskowski RA, MacArthur MW, Moss DS & Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structure J Appl Crystallogr 26, 283–291 32 Rodriguez R, Chinea G, Lopez N, Pons T & Vriend G (1998) Homology modeling, model and software evaluation: three related resources Bioinformatics 14, 523–528 33 Barlow DJ & Thornton JM (1983) Ion-pairs in proteins J Mol Biol 168, 867–885 34 Nicholls A, Sharp KA & Honig B (1991) Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons Proteins 11, 281–296 35 Thompson JD, Higgins DG & Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 22, 4673–4680 Supplementary material The following supplementary material is available online: Fig S1 Representative portion of the final rAweighted 2|Fo| – |Fc| electron-density map of A fulgidus L-aspDH with bound NAD contoured at the 1r level (blue), together with a fitted model of NAD shown as a stick model in magenta Fig S2 The inter- and intrasubunit aromatic pairs in T maritima L-aspDH (A) and A fulgidus L-aspDH (B) The regions of the intersubunit aromatic pair and the largest aromatic pair network are indicated by a FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS K Yoneda et al circle and a broken-line circle, respectively Residues belonging to A and B molecules are shown in green and magenta, respectively This material is available as part of the online article from http://www.blackwell-synergy.com Crystal structure of L -aspDH from A fulgidus Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4325 ... nicotinamide ring of NAD Based on the structure of citrate, we modeled the l-aspartate molecule into the active site of A fulgidus l-aspDH (Fig 4B) and then minimized the energy of the complex using... The crystal structure of a hyperthermophilic archaeal TATA-box binding protein J Mol Biol 264, 1072–1084 Lim JH, Yu YG, Han YS, Cho S, Ahn BY, Kim SH & Cho Y (1997) The crystal structure of an... thermostability of each enzyme are discussed Finally, we describe a substrate-induced conforma4316 tional change in the ternary complex of A fulgidus l-aspDH Results and Discussion Overall structure The structure