Crystal structure of a putative glutamine amido transferase (TM1158) from Thermotoga maritima at 1.7 Å resolution (2004)

The TM1158 gene of Thermotoga maritima encodes a putative glutamine amidotransferase (EC 2.6.1.15) with a molecular weight of 26,901 Da and a calculated isoelectric point of 5.8. Glutamine amidotransferases (GATases) transfer the amide nitrogen of glutamine to a variety of substrates.1 The chemical transformation is carried out within two active sites, which are located in the glutaminase and synthase subunits of either fused or separate polypeptide chains.2 Here, we report the crystal structure of the glutaminase subunit, TM1158, determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).3 The structure of TM1158 [Fig. 1(A)] was determined to 1.7 Å resolution using the multiple-wavelength anomalous dispersion (MAD) technique. Data collection, model, and refinement statistics are summarized in Table I. The final model includes one protein molecule in the asymmetric unit (residues 1–227, plus 3 N-terminal His tag residues), 9 sulphates, and 382 water molecules. The Matthews' coefficient (Vm) for TM1158 is 2.55 Å3/Da and the estimated solvent content is 51.3%. The Ramachandran plot produced by PROCHECK 3.44 shows that 92% of the residues are in the most favored regions, and 8% in additional allowed regions. With the exception of the active site cysteine C88, no residues lie in the generously allowed or disallowed regions. (A) Ribbon diagram of T. maritima TM1158 color coded from N-terminus (blue) to C-terminus (red) showing the domain organization. The location of the active site triad (C88–H173–E175) is indicated with an arrow. C88 is present as sulfonic acid (OCS) with the three oxygen atoms rendered in ball and stick (red). Other atoms are indicated as follows: carbon (gray), nitrogen (blue), oxygen (red), and sulfur (orange). The α-helices (H1–H9), as well as the β-strands (β1–β11) are labeled. (B) Diagram showing the secondary structure elements in TM1158 superimposed on its primary sequence. β-hairpins are depicted in red. The sequence corresponding to the helical insert is marked with a black line. The TM1158 monomer consists of a single polypeptide chain of 227 amino acids composed of 9 helices (6 α-helices and 3 310-helices), and 11 β-strands (Fig. 1). The total α-helix, 310-helix, and β-strand content is 32.3%, 3.9%, and 23.1%, respectively. The TM1158 structure has a three-layer α–β–α flavodoxin-like fold,5 with a mostly parallel β-sheet, where the first 4 parallel β-strands (β1, β2, β3, β4) with a 2134 order are followed by an additional β-domain (β5, β6, β7, β8, β9, β10, β11) that contributes three antiparallel β-strands, β11, β10, and β9, to the main β-sheet [Fig. 1(A)]. This structure is typical for glutaminase subunits of other Class 1 GATases,6 but contains an additional α-helical “hairpin” within residues 176–210, corresponding to helices H7–H9, inserted at the C-terminus [Figs. 1 and 2(A)]. TM1158 also contains a catalytic Cys–His–Glu triad (C88–H173–E175) with a similar conformation to previously determined protein structures of this type6 [Fig. 2(B)]. The active-site Cys-88 is strained (φ = 67.6°, ψ = −117.7°) in a normally disallowed region of the Ramachandran plot,4 but is a characteristic feature in glutaminase structures.7 Here, the active site cysteine is oxidized to cysteine sulfonic acid (OCS) resulting in a different rotamer, as compared to Cys-85 in the Trpg subunit of anthranilate synthase,7 most likely due to the steric hindrance of the sulfonic acid moiety. A sulphate molecule is bound close to the active site. Analogy with other GATases strongly suggests that a monomer is the biologically-relevant form. (A) Superposition of TM1158 (blue) and the Trpg-subunit (gray) of anthranilate synthase from S. solfataricus (PDB: 1QDL). The “helical” insert is marked with a circle and the active site residues for TM1158 are shown in ball-and-stick. (B) Close-up view of the active site comparison. Residues for the two active site triads (TM1158 blue, anthralinate synthase gray) are shown in ball-and-stick. The hydrogen-bond distances between catalytic triad residues are indicated for TM1158 with anthralinate synthase in brackets. A structural similarity search, performed with the coordinates of TM1158 using the DALI server8 indicates that the closest structural homologue is the glutaminase subunit (Trpg) of anthranilate synthase from Sulfolobus solfataricus [Protein Data Bank (PDB): 1QDL].7 The root-mean-square deviation (RMSD) between TM1158 and the Trpg subunit of 1QDL is 2.1 Å over 184 aligned residues (Fig. 2). Sequence alignment of these superimposed structures shows 18% identity. According to fold and function assignment system (FFAS),9 several structural homologues are present in the Thermotoga proteome: TM1820, GMP synthase (guaA) with 24% sequence identity; TM1245, phosphoribosylformylglycinamidine synthase I (purQ) with 17% sequence identity; TM0141, anthranilate synthase (Trpg) with 16% sequence identity; and TM1038, amidotransferase (hisH) with 13% sequence identity. All of these proteins contain the catalytic Cys–His–Glu triad and are involved in amidotransferase reactions. Models for a number of TM1158 orthologs and paralogs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=tm1158. The structure reported here represents a putative glutaminase whose structure has been determined by X-ray crystallography using the MAD method. In comparison to glutaminase subunits of known GATases, TM1158 contains a novel feature consisting of an α-helical “hairpin,” residues 176–210, inserted before the C-terminal α-helix. The information reported here will provide valuable insights into the determinants for catalysis, substrate specificity, and thermal stability of this protein family. TM1158 (TIGR: TM1158; Swiss-prot: Q9X0P2) was amplified by polymerase chain reaction (PCR) from T. maritima strain MSB8 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of TM1158. The PCR product was cloned into plasmid pMH1, which encodes an expression and a purification tag corresponding to residues MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in 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 cell debris pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a metal chelate affinity resin (Amersham Biosciences) previously charged with nickel and equilibrated with Equilibration Buffer (50 mM potassium phosphate pH 7.8, 0.25 mM TCEP, 10% v/v glycerol, 300 mM NaCl) containing 20 mM imidazole. The resin was washed with Equilibration Buffer containing 40 mM imidazole, and the protein eluted with Elution Buffer (20 mM Tris pH 7.9, 10% glycerol, 0.25 mM TCEP, 300 mM imidazole). 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) previously equilibrated with the same buffer. The flow-through fractions were pooled, buffer exchanged into size exclusion chromatography (SEC) Buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP), and concentrated for crystallization assays by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method10 with standard JCSG crystallization protocols.3 The crystallization solution contained 2.4 M (NH4)2SO4 and 0.1 M bicine at pH 9.0. The crystals were indexed in the hexagonal space group P6522 (Table I). Native and multiwavelength anomalous diffraction data were collected at the Stanford Synchroton Radiation Laboratory (Stanford, CA) on beamlines 19BM and 11-1 at multiple wavelengths using the BLU-ICE11 data collection environment (Table I). All data sets were collected at 100 K using a Quantum 4 charge-coupled device (CCD) detector. Data were integrated and reduced using Mosflm,12 then scaled with the program SCALA from the CCP4 suite.13 Data statistics are summarized in Table I. The structure was determined using the software packages SnB,14 the CCP4 suite,13 and RESOLVE.15 Since initially only 2.8 Å data were available, the first model was constructed based on anthranilate synthase from S. solfataricus (PDB: 1QDL)7 superposed on the auto-traced fragments from RESOLVE. The structure was then refined to a resolution of 1.7 Å using REFMAC5.13 The asymmetric unit contains one molecule. Refinement statistics are summarized in Table I. The final model contains the entire protein structure (residues 1–227) plus residues His −2, −1, and 0 from the purification tag. The electron density maps indicate that the active site cysteine is oxidized to cysteine sulfonic acid (OCS), as observed in cysteine proteinase structures.16 The following residues have side-chains with alternative conformations: 61E, 75E, 121D, 127E, 133R, 163V, and 219S. Side-chains for residues K68 and R200 were disordered and, therefore, excluded from the model. Analysis of the stereochemical quality of the models was accomplished using the JCSG Validation Central suite, which integrates seven validation tools: Procheck 3.5.4, SFcheck 4.0, Prove 2.5.1, ERRAT, WASP, DDQ 2.0, and Whatcheck. The Validation Central suite is accessible at http://www.jcsg.org. Atomic coordinates of the final model and experimental structure factors of TM1158 have been deposited with the PDB and are accessible under the code 1o1y. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, 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).