Aspartate Dehydrogenase, a Novel Enzyme Identified from Structural and Functional Studies of TM1643

The open reading frame TM1643 of Thermotoga maritima belongs to a large family of proteins, with homologues in bacteria, archaea, and eukaryotes. TM1643 is found in an operon with two other genes that encode enzymes involved in the biosynthesis of NAD. In several bacteria, the gene in the position occupied by TM1643 encodes an aspartate oxidase (NadB), which synthesizes iminoaspartate as a substrate for NadA, the next enzyme in the pathway. The amino acid sequence of TM1643 does not share any recognizable homology with aspartate oxidase or with other proteins of known functions or structures. To help define the biological functions of TM1643, we determined its crystal structure at 2.6Å resolution and performed a series of screens for enzymatic function. The structure reveals the presence of an N-terminal Rossmann fold domain with a bound NAD+ cofactor and a C-terminal α+β domain. The structural information suggests that TM1643 may be a dehydrogenase and the active site of the enzyme is located at the interface between the two domains. The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward l-aspartate but no aspartate oxidase activity. The product of the aspartate dehydrogenase activity is also iminoaspartate. Therefore, our studies demonstrate that two different enzymes, an oxidase and a dehydrogenase, may have evolved to catalyze the first step of NAD biosynthesis in prokaryotes. TM1643 establishes a new class of amino acid dehydrogenases. The open reading frame TM1643 of Thermotoga maritima belongs to a large family of proteins, with homologues in bacteria, archaea, and eukaryotes. TM1643 is found in an operon with two other genes that encode enzymes involved in the biosynthesis of NAD. In several bacteria, the gene in the position occupied by TM1643 encodes an aspartate oxidase (NadB), which synthesizes iminoaspartate as a substrate for NadA, the next enzyme in the pathway. The amino acid sequence of TM1643 does not share any recognizable homology with aspartate oxidase or with other proteins of known functions or structures. To help define the biological functions of TM1643, we determined its crystal structure at 2.6Å resolution and performed a series of screens for enzymatic function. The structure reveals the presence of an N-terminal Rossmann fold domain with a bound NAD+ cofactor and a C-terminal α+β domain. The structural information suggests that TM1643 may be a dehydrogenase and the active site of the enzyme is located at the interface between the two domains. The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward l-aspartate but no aspartate oxidase activity. The product of the aspartate dehydrogenase activity is also iminoaspartate. Therefore, our studies demonstrate that two different enzymes, an oxidase and a dehydrogenase, may have evolved to catalyze the first step of NAD biosynthesis in prokaryotes. TM1643 establishes a new class of amino acid dehydrogenases. open reading frame aspartate semialdehyde dehydrogenase The open reading frame (ORF)1 TM1643 was identified in the genome of the hyperthermophilic bacterium Thermotoga maritima (1Nelson K.E. Clayton R.A. Gill S.R. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Nelson W.C. Ketchum K.A. McDonald L. Utterback T.R. Malek J.A. Linher K.D. Garrett M.M. Stewart A.M. Cotton M.D. Pratt M.S. Phillips C.A. Richardson D. Heidelberg J. Sutton G.G. Fleischmann R.D. Eisen J.A. Fraser C.M. et al.Nature. 1999; 399: 323-329Google Scholar). This ORF encodes a soluble, 241-residue protein that is conserved among a large number of organisms, includingCaenorhabditis elegans and humans (Fig.1). Currently, there are more than 15 homologs of this protein in the data base, suggesting it may have an important, conserved function in these living systems. However, the function of this protein cannot be deduced from its sequence, because it does not share any recognizable similarity to other proteins of known function or structure. TM1643 is predicted to be the first gene in a three-gene operon in T. maritima (1Nelson K.E. Clayton R.A. Gill S.R. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Nelson W.C. Ketchum K.A. McDonald L. Utterback T.R. Malek J.A. Linher K.D. Garrett M.M. Stewart A.M. Cotton M.D. Pratt M.S. Phillips C.A. Richardson D. Heidelberg J. Sutton G.G. Fleischmann R.D. Eisen J.A. Fraser C.M. et al.Nature. 1999; 399: 323-329Google Scholar). The second and third genes of this operon have sequence homology to NadA and NadC in Escherichia coli, respectively, which encode proteins that catalyze thede novo biosynthesis of NAD from l-aspartate. In prokaryotes, the first reaction in this pathway is catalyzed by the FAD-containing l-aspartate oxidase (NadB), which oxidizesl-aspartate to iminoaspartate using oxygen or fumarate as electron acceptors (2Nasu S. Wicks F.D. Gholson R.K. J. Biol. Chem. 1982; 257: 626-632Google Scholar) (Fig. 2). Iminoaspartate is then condensed with dihydroxyacetone phosphate to produce quinolinate, a reaction catalyzed by NadA (quinolinate synthetase A). The third reaction in this pathway is catalyzed by NadC (quinolinate phosphoribosyltransferase). These three genes,nadB-nadA-nadC, make an operon in thermophilic bacteria (Pyrococcus, Sulfolobus) and inBacillus. Although the TM1643 gene occupies the equivalent position of NadB in the operon, it does not share any recognizable sequence similarity to NadB. To help define the functional role of this ORF, we have determined its crystal structure at 2.6 Å resolution as part of our structural genomics effort. The structure revealed that TM1643 may be an NAD(P)+-dependent oxidoreductase. Based on the structural information, our enzymatic characterization of this protein showed that it is a dehydrogenase strictly specific forl-aspartate. Our structural and functional studies, together with the position of TM1643 in the Nad operon, therefore suggest that TM1643 and its homologues are aspartate dehydrogenases that catalyze the first step in NAD biosynthesis from aspartate. These results establish a new class of amino acid dehydrogenases. Both aspartate oxidase and aspartate dehydrogenase produce iminoaspartate from the oxidation of l-aspartate (Fig. 2). The difference between them is that the former catalyzes the reduction of FAD (and ultimately oxygen or fumarate), whereas the latter catalyzes the reduction of NAD(P). Recombinant protein was expressed with a hexa-histidine fusion tag at the N terminus inE. coli BL21 (DE3). The cells were lysed in a buffer containing 50 mm Hepes (pH 7.5), 500 mm NaCl, 5% (v/v) glycerol, 1 mm phenylmethylsulfonyl fluoride, and 0.5 mm benzamidine. The soluble recombinant protein was bound to Ni+2 affinity resin and eluted in a buffer containing 50 mm Hepes, pH 7.5, 500 mm NaCl, 5% (v/v) glycerol, and 250 mmimidazole. The purified protein was dialyzed extensively against a buffer containing 10 mm Hepes and 500 mm NaCl, concentrated to 20 mg/ml, and stored at 4 °C. Crystals were grown at room temperature using the hanging-drop vapor diffusion method. The reservoir solution contained 0.2 m KH2PO4, 22% polyethylene glycol 1500, and 1 mm EDTA. Crystals were transferred to a cryoprotectant solution containing 25% ethylene glycol and flash-frozen in liquid nitrogen for data collection at 100 K. A single-wavelength anomalous diffraction (SAD) data set to 2.6 Å resolution was collected at the National Synchrotron Light Source beam lines X4A. The crystal belongs to space groupP41212, with cell parameters ofa = b = 63.2 Å and c = 125.1 Å. The diffraction images were processed with the HKL package (Table I) (3Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326PubMed Google Scholar).Table ISummary of crystallographic informationMaximum resolution (Å)2.6Number of observations87,706R merge(%)aRmerge=∑h∑i‖Ihi−〈Ih〉‖/∑h∑iIhi6.7Resolution range for refinement10–2.6Number of reflections8,163Completeness (%)99R/freeR factor(%)bR=∑h‖Fho−Fhc‖/∑hFho22.6/29.1r.m.s. deviation in bond lengths (Å)0.007r.m.s. deviation in bond angles (°)1.3a Rmerge=∑h∑i‖Ihi−〈Ih〉‖/∑h∑iIhib R=∑h‖Fho−Fhc‖/∑hFho Open table in a new tab The Se sites were located with the SnB program based on the anomalous differences (4Weeks C.M. Miller R. J. Appl. Crystallogr. 1999; 32: 120-124Google Scholar). Reflection phases were calculated and solvent-flattened with the program Solve (5Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Google Scholar). The atomic model was built into the electron density map with the program O (6Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Google Scholar), and the structure refinement was carried out with the program CNS (7Brunger 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: 905-921Google Scholar). The crystallographic information is summarized in Table I. Dehydrogenase activity against pools of different substrates (20 amino acids, 9 organic acids, or 8 alcohols) was measured spectrophotometrically by following the increase of absorbance at 340 nm. General assays were performed at 70 °C in a reaction mixture containing 100 mm Tricine buffer (pH 8.5), 0.5 mm NADP+, 0.5 mmNAD+, and 5 mm of the substrate mixture. Dehydrogenase activity with individual amino acids as substrates was measured in a reaction mixture containing 50 mmdiethanolamine buffer (pH 9.8), 1 mm NADP+ (or NAD+), and 5 mm of the amino acid. Asp dehydrogenase activity in the direction of reductive amination (NADPH or NADH oxidation) was monitored at 70 °C as the decrease at 340 nm in a 1-ml mixture containing 50 mm Tris-HCl (pH 8.0), 100 mm (NH4)2SO4, 0.1 mm EDTA, 0.15 mm NADPH (or NADH), 5 mm oxaloacetate, and appropriate amount of enzyme. The products of the reaction with l-aspartic acid were detected using enzymatic assays. NH4+ was measured with glutamate dehydrogenase (8Bergmeyer H.U. Methods Enzymol. 1985; 8: 454-461Google Scholar). Pyruvate was measured with lactate dehydrogenase (9Bergmeyer H.U. Methods Enzymol. 1985; 6: 570-577Google Scholar) and with alanine dehydrogenase (10Yoshida A. Freese E. Methods Enzymol. 1970; 17: 176-181Google Scholar). The spectrophotometric assay for l-Asp oxidase activity was carried out using the horseradish peroxidase-coupled method by following the oxidation of o-dianisidine at 460 nm (11Tedeschi G. Negri A. Mortarino M. Ceciliani F. Simonic T. Faotto L. Ronchi S. Eur. J. Biochem. 1996; 239: 427-433Google Scholar). The aspartate semialdehyde dehydrogenase (ASA-DH) activity in the biosynthetic direction (the synthesis ofl-aspartate-β-semialdehyde) was examined by using the previously described aspartate kinase-coupled method (12Angeles T.S. Viola R.E. Arch. Biochem. Biophys. 1990; 283: 96-101Google Scholar). The ASA-DH activity in the phosphorylating direction was assayed at 70 °C with aspartate semialdehyde as a substrate (a kind gift from Dr. Wright) by following the increase in the absorbance at 340 nm as described previously (13Karsten W.E. Viola R.E. Biochim. Biophys. Acta. 1991; 1077: 209-219Google Scholar). The inositol-1-phosphate synthase activity was examined as previously described (14Wong Y.H. Mauck L.A. Sherman W.R. Methods Enzymol. 1982; 90: 309-314Google Scholar). The crystal structure of the TM1643 ORF of T. maritima in complex with NAD+has been determined at 2.6 Å resolution by the seleno-methionyl SAD method (15Hendrickson W.A. Science. 1991; 254: 51-58Google Scholar). The current R factor of the model is 22.6%. The crystallographic information is summarized in TableI. The atomic coordinates have been deposited at the Protein Data Bank with the accession code 1H2H. While our structure determination was in progress, another research group deposited the structure of this protein in the PDB (entry 1J5P). The rms distance for all the equivalent Cα atoms of the two structures is 0.3 Å. The structure of TM1643 contains two domains. The backbone fold of the N-terminal domain (residues 1–105) is essentially the same as that of the canonical Rossmann fold, with a central six-stranded parallel β-sheet (Fig. 3 A). The NAD+ molecule is associated with this domain at a position that is equivalent to the binding site in other Rossmann folds. This structural observation immediately suggests that TM1643 may be an oxidoreductase. The C-terminal domain (residues 113–241) has an open-faced sandwich structure with a five-stranded mixed β-sheet with three helices on one side (Fig. 3 A). The nicotinamide ring of NAD+ is located at the interface between the two domains, indicating that this likely is the location of the active site of this enzyme (Fig. 3 A). The C-terminal domain also helps to mediate the dimerization of the protein. The β-sheets in the C-terminal domains of the two monomers are arranged in a side-by-side fashion, and this creates a ten-stranded β-sheet in the dimer (Fig. 3 B). In addition, the C-terminal residues of one monomer form a short β-strand, and it extends the six-stranded β-sheet in the N-terminal domain of the other monomer (Fig. 3 B). Our structural observation suggests that TM1643 is likely to exist as dimers in solution. Searches with the program Dali against the data base of known protein structures (16Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Google Scholar) revealed that TM1643 has weak structural homology to several other NAD(P)+–dependent oxidoreductases, including inositol 1-phosphate synthase (17Norman R.A. McAlister M.S.B. Murray-Rust J. Movahedzadeh F. Stoker N.G. McDonald N.Q. Structure. 2002; 10: 393-402Google Scholar), dihydrodipicolinate reductase (18Scapin G. Blanchard J.S. Sacchettini J.C. Biochemistry. 1995; 34: 3502-3512Google Scholar), and ASA-DH (19Hadfield A. Shammas C. Kryger G. Ringe D. Petsko G.A. Ouyang J. Viola R.E. Biochemistry. 2001; 40: 14475-14483Google Scholar). The amino acid sequence identity among the structurally equivalent residues is below 15%, underscoring the lack of sequence homology between TM1643 and other proteins. As could be expected, this structural homology is stronger in the N-terminal Rossmann fold domain. The core of the C-terminal β-sheet is similar among these structures, although there are also significant variations such as the presence of additional strand(s) in the sheet. Overall, the structural homology strongly supports the notion that TM1643 is also an oxidoreductase (dehydrogenase). In addition, open and closed forms of the structures have been observed from studies on these other enzymes, with changes in the relative positioning of the two domains. Our structure of TM1643 appears to be in an open form. The active site of TM1643 is located at the interface between the N- and C-terminal domains of the monomer. Residues in this active site come from strands β7 and β10, helices αA, αF, and αG, the β5-αC loop and the αE-αF loop, and finally the linker between the two domains (residues 106–112) (Fig. 4 A). They are generally well conserved among this family of proteins (Fig. 1). There is a clear depression on the surface of the protein, delineating the region for substrate binding and catalysis (Fig. 4 B). This active site region is open to the solvent in the current structure (Fig. 4 B). It is expected that the active site will be shielded from the solvent in the closed form of the enzyme when the substrate is bound. Our structural analysis showed that the His-193 residue may function as the general acid/base in the catalysis by these enzymes. This residue is strictly conserved among all family members and may be located near the C4 position of the nicotinamide ring in the closed form of the enzyme. In support of this observation, the His-193 residue is structurally equivalent to the catalytic His residue in ASA-DH (19Hadfield A. Shammas C. Kryger G. Ringe D. Petsko G.A. Ouyang J. Viola R.E. Biochemistry. 2001; 40: 14475-14483Google Scholar). A short loop of the enzyme, residues 212–219 (just prior to helix αG, Figs. 2 A and 3 A), is disordered in our current structure. These residues may be located in the active site, helping form a flap over the active site when the substrate is bound. Although TM1643 is located in the position corresponding to the aspartate oxidase gene (nadB) of the Nad operons, it does not share any sequence or structural homology with l-aspartate oxidases. Furthermore, purified TM1643 showed no evidence for the presence of an FAD cofactor, and the enzyme has no l-aspartate oxidase activity (data not shown). It is therefore highly unlikely that TM1643 is an aspartate oxidase. TM1643 shows structural homology to ASA-DH. However, it is unlikely to be an ASA-DH because its active site lacks the Cys residue required for the catalysis by ASA-DH (19Hadfield A. Shammas C. Kryger G. Ringe D. Petsko G.A. Ouyang J. Viola R.E. Biochemistry. 2001; 40: 14475-14483Google Scholar). Our enzymatic studies with purified TM1643 showed no ASA-DH activity in either the forward or the reverse reaction. In addition, purified TM1643 has no homoserine dehydrogenase or inositol-1-phosphate synthase activity, the latter enzyme being a structural homolog of TM1643. With the information obtained from the structural studies, and from the analysis of the operon sequence conservation, we next performed enzymatic screens to explore various compounds as potential substrates for the presumed TM1643 dehydrogenase activity. The structural information suggested that TM1643 would likely possess dehydrogenase activity. To identify substrate(s) for TM1643, we designed general dehydrogenase screens using a mixture of NAD and NADP as the electron acceptors and three mixtures of substrates (amino acids, organic acids, or alcohols) as electron donors. Significant dehydrogenase activity was observed only with the pool of amino acids. When tested individually as electron donors, TM1643 showed robust dehydrogenase activity towardl-aspartate. No activity was found withd-aspartate; the enzyme is strictly specific forl-aspartate. l-glutamate and asparagine are not substrates of this enzyme. Purified TM1643 showed classical Michaelis-Menten kinetics, and linear double-reciprocal plots were obtained with all three substrates tested,l-aspartate, NADP, and NAD (Fig.5, A and B). The TM1643 kinetic parameters are presented in TableII. Interestingly, theK m for l-Asp was ∼20 times lower in the presence of NAD than in the presence of NADP (Fig. 5 Band Table II). Noting the lower K m for NAD andl-aspartate, it is likely that the TM1643 dehydrogenase uses NAD as the cofactor for the oxidation of aspartate in vivo. This is also consistent with our observation that NAD was co-purified and co-crystallized with TM1643 (Fig. 3 A).Table IISteady-state kinetic parameters for TM1643 and E. coli aspartate oxidaseVariable substrateSaturated substrateK mk catk cat/K mmMs −1M −1 s −1TM1643l-AspNADP1.20 ± 0.054.9 ± 0.094.1 × 103l-AspNAD0.067 ± ± × ± ± × ± ± × coli aspartate parameters for E. coli aspartate oxidase with or fumarate as electron acceptors × × parameters for E. coli aspartate oxidase with or fumarate as electron acceptors (11Tedeschi G. Negri A. Mortarino M. Ceciliani F. Simonic T. Faotto L. Ronchi S. Eur. J. Biochem. 1996; 239: 427-433Google Scholar). Open table in a new tab The l-aspartate dehydrogenase activity of TM1643 was not by the of or and NH4+ were with K = ± mm and ± the reaction catalyzed by aspartate aspartate dehydrogenase is also expected to produce iminoaspartate from the oxidation of l-aspartate (Fig. 2). In the iminoaspartate product is not and its will produce and (Fig. 2). examined the products by the reaction dehydrogenase NADPH and was detected in to the NADH (data not shown). were to oxaloacetate, because it is the that we (pH and 70 is likely to to and we were to identify the of by using two enzymatic assays (data not shown). could also demonstrate the reverse reductive amination of by purified TM1643 at 70 °C and pH using reaction previously described for glutamate dehydrogenase (8Bergmeyer H.U. Methods Enzymol. 1985; 8: 454-461Google Scholar). In this reverse NADPH and NADH were as electron donors. Our structural and enzymatic studies demonstrate that TM1643 is a amino acid dehydrogenase that is strictly specific for l-aspartate. This a its functional role in T. T. maritima contains a well glutamate dehydrogenase that a role in the and nitrogen in all Therefore, it is unlikely that the TM1643 aspartate dehydrogenase the same role in T. TM1643 does not share structural similarity to the of and dehydrogenases Rice J. Biol. Chem. 1997; Scholar) and establishes a new class of amino acid dehydrogenases. These other enzymes catalyze the of the amino acid substrates Biochemistry. Biochemistry. 2002; Scholar) (Fig. 2). In TM1643 is part of the Nad operon in T. and our analysis not the presence of an l-aspartate oxidase homolog therefore that in this TM1643 catalyzes the first reaction of novo biosynthesis of NAD from and it iminoaspartate required for this pathway. The of an enzyme complex between TM1643 and NadA, the next enzyme of the may the of this product to the NadA active and for help with data collection at and for the Viola for and and for help with ASA-DH

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