Crystal structure of a formiminotetrahydrofolate cyclodeaminase (TM1560) from Thermotoga maritima at 2.80 Å resolution reveals a new fold (2005)

The TM1560 gene of Thermotoga maritima encodes a formiminotetrahydrofolate cyclodeaminase (FTCD_CD, EC 4.3.1.4), with a molecular weight of 22,581 Da (residues 1–202) and a calculated isoelectric point of 5.0.1 This enzyme is part of the folate metabolic pathway and catalyzes the cyclization of the formimino group of N5-formimidoyltetrahydrofolate, yielding N5,N10-methenyltetrahydrofolate and ammonia2 [Fig. 1(A)]. In eukaryotes, it is fused to glutamate formiminotransferase (FTCD_FT, EC 2.1.2.5), which catalyzes the previous step in the pathway (from tetrahydrofolate to N5-formiminotetrahydrofolate) to assemble the bifunctional enzyme, formiminotransferase-cyclodeaminase (FTCD; EC 2.1.2.5, EC 4.3.1.4).3 In Thermotoga maritima, FTCD_FT is encoded by a separate gene (TM0843). A genome context analysis reveals that close homologues of TM1560 form clusters with enzymes involved in folate metabolism in various bacterial genomes. For example, the cluster in Clostridium tetani and Thermoplasma volcani contains genes encoding glutamate formiminotransferase [Enzyme Commission (EC) 2.1.2.5) and formate-tetrahydrofolate ligase (EC 6.3.4.3). (A) Scheme for the reaction carried out by FTCD_CD (EC 4.3.1.4), which catalyzes the cyclization of the formimino group of N5-formimidoyltetrahydrofolate, yielding N5,N10-methenyltetrahydrofolate and ammonia. (B) Crystal structure of TM1560. Ribbon diagram of T. maritima FTCD_CD color-coded from N-terminus (blue) to C-terminus (red) showing the domain organization. Helices H1–H6 are indicated. (C) The TM1560 dimer view is shown along (above) and normal (below) to its two-fold axis. (D) Diagram showing the secondary structure elements in TM1560 superimposed on its primary sequence. The α-helices, 310-helices, β-bulges, and γ-turns are indicated. Disordered regions are depicted by a dashed line with the corresponding sequence in brackets. Here, we report the crystal structure of FTCD_CD determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).4 The crystal structure of FTCD_CD [Fig. 1(A)] was determined to 2.80 Å resolution using the single-wavelength anomalous dispersion (SAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes 2 protein monomers (residues 2–201 for chain A and residues 3–199 for chain B). The Matthews coefficient (Vm)7 for TM1560 is 2.84 Å3/Da and the estimated solvent content is 56.4%. The Ramachandran plot, produced by MolProbity,8 shows that 98.5% and 1.5% of the residues are in favored regions and additionally allowed regions, respectively. The final model of the TM1560 monomer consists of 6 α-helices (H1–H6) with three 310-helical segments (H3′, H5′, H6′), 8 β-turns, and 1 γ-turn [Fig. 1(B and D)]. The total α-helical and 310-helical content is 76.5% and 3.5%, respectively. The TM1560 monomer is composed of 6 α-helices arranged in an up-and-down helical bundle [Fig. 1(B)]. The crystallographic packing in the TM1560 structure suggests that a dimer is the biologically relevant oligomeric form with a buried surface area of 2301 Å2 per monomer. The dimer is formed through interactions of helices H1, H2, H5, and H6 [Fig. 1(C)] and contains 68% nonpolar atoms and 15 hydrogen bonds. A structural similarity search, performed with the coordinates of TM1560 using the DALI server,9 finds no structural homologues for the whole domain indicating that TM1560 is a new fold [Fig. 2(A)]. The closest structural homologue, the invertase inhibitor Nt-Cif from tobacco [Protein Data Bank (PDB) code: 1rj1],10 shows some similarity in the arrangement of helices H2–H5, but the helices in Nt-Cif are much shorter [Fig. 2(A)]. The root-mean-square deviation (RMSD) for this structural alignment is 3.2 Å over 109 aligned residues with 8% sequence identity. (A) Superposition of TM1560 (gray) and invertase inhibitor Nt-Cif from Tobacco (PDB code: 1rj1, magenta). (B) The putative active site pocket in surface representation. Residues are colored according to sequence conservation, where green is conserved and white is nonconserved. The manually docked ligand N5, N10-methenyltetrahydrofolate (MTHF) is shown in stick representation (yellow). (C) Same orientation as (B), but shown in ribbon representation, with conserved residues in the putative active site of TM1560 and the manually docked MTHF ligand in stick representation. Monomers 1 and 2 in the dimer are shown in blue and gray, respectively. The location of the TM1560 active site and its reaction mechanism are unknown. A map of conserved residues from an alignment of available FTCD_CD sequences reveals 2 identical clusters of conserved residues in the dimer interface. These clusters are located in a deep pocket (surface area: ∼800 Å2, volume: ∼1250 Å3) that is large enough to accommodate the product (N5, N10-methenyltetrahydrofolate, MTHF), which indicates a possible location of the FTCD_CD active site [Fig. 2(B and C)]. A similar placement of active sites in the dimer interface is also seen in glutamate formiminotransferase FTCD_FT.3 According to our modeling studies [Fig. 2(B and C)], the conserved residues can be divided into 2 groups according to their functional roles. The first group of residues (M41, F45, K49, K50, N131, N133, S136, and D137) from monomer 1 may be responsible for binding the p-aminobenzoyl portion of the ligand and catalysis. The second group (P20, G23, G24, G25, D80, F84, P109, N155, I158, and N159) from monomer 2, is located at the bottom of the groove, most likely binds and stabilizes the tetrahydropteridine ring of the substrate. This binding mode would put the strictly conserved aspartate, D137, close to the site where the deamination reaction occurs. Because aspartates or glutamates frequently serve as catalytic residues in deaminase reactions,11 we propose that D137 acts as the catalytic residue in FTCD_CD, and may extract a proton from the formiminogroup of the substrate to facilitate its deamination and subsequent cyclization. According to Fold and Function Assignment System (FFAS),12 the subfamily including TM1560 currently has 28 homologous sequences in all kingdoms of life. Models for TM1560 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM1560. The TM1560 structure reported here represents a formiminotetrahydrofolate cyclodeaminase from T. maritima, whose structure has been determined by X-ray crystallography using the SAD method. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the catalytic mechanism and functional determinants of this protein. Protein production and crystallization: Formiminotetrahydrofolate cyclodeaminase from T. maritima (TIGR: TM1560, Swiss-Prot: Q9X1P6) was amplified by polymerase chain reaction (PCR) from genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 250 μg/mL. Bacteria were lysed by sonication after a freeze-thaw procedure in Lysis Buffer [50 mM Tris, pH 7.9, 50 mM NaCl, 0.25 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)], and the cell debris was pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a nickel-resin (Amersham Biosciences) pre-equilibrated with Equilibration Buffer [50 mM potassium phosphate, pH 7.8, 300 mM NaCl, 10% (v/v) glycerol, 0.25 mM TCEP] containing 20 mM imidazole. The nickel-resin was washed with Equilibration Buffer containing 40 mM imidazole, and the protein was eluted with Elution Buffer [20 mM Tris, pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP]. Buffer exchange was performed to remove imidazole from the eluate, and the protein in Buffer Q [20 mM Tris, pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl was applied to a Resource Q column (Amersham Biosciences) pre-equilibrated with the same buffer. The protein was eluted using a linear gradient of 50 to 500 mM NaCl in Buffer Q. The appropriate fractions were pooled, buffer exchanged in Crystallization Buffer [20 mM Tris, pH 7.9, 150 mM NaCl, 0.25 mM TCEP] and concentrated to 12 mg/mL for crystallization by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method,13 with standard JCSG crystallization protocols.4 The crystallization reagent contained 5% polyethylene glycol 6000 (PEG-6000), 0.1 M citric acid at pH 5.0, and 35% glycerol (final concentration) was included as a cryoprotectant. The crystals were indexed in the orthorhombic space group P212121 (Table I). Data collection: Anomalous diffraction data were collected at the Advanced Light Source (ALS; Berkeley, CA) on beamline 8.2.2 at the wavelength corresponding to the peak (λ1) in a selenium SAD experiment (Table I). The data sets were collected at 100 K using a Quantum 315 charge-coupled device (CCD) detector. Data were integrated and reduced using MOSFLM14 and then scaled with the program SCALA from the CCP4 suite.5 Data statistics are summarized in Table I. Structure solution and refinement: The structure was determined by using the CCP4 suite5 and SOLVE/RESOLVE.15 Structure refinement was performed using REFMAC55 and Xfit.16 Refinement statistics are summarized in Table I. The final model includes 2 protein monomers (residues 2–201, 3–199) in the asymmetric unit. No electron density was observed for residues 1 and 202 in chain A, residues 1, 2, 200–202 in chain B, the side-chain atoms of R5, K19, K48, K50, E68, K89, Y91, K92, K170, and K192, and the rest of the expression and purification tags. Validation and deposition: Analysis of the stereochemical quality of the model was accomplished using the AutoDepInputTool (http://deposit.pdb.org/adit/), MolProbity,8 SFcheck 4.0,5 and WHATIF 5.0.17 Protein quaternary structure analysis used the PQS server (http://pqs.ebi.ac.uk/). Figure 1(B) was adapted from an analysis using PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/), and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of TM1560 have been deposited within the PDB and are accessible under the code 1o5h. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL) and the ALS. The SSRL is a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.