Crystal structure of a type II quinolic acid phosphoribosyltransferase (TM1645) from Thermotoga maritima at 2.50 Å resolution (2004)

The TM1645 gene of Thermotoga maritima encodes a type II quinolic acid phosphoribosyltransferase (QAPRTase, NadC, EC 2.4.2.19), with a molecular weight of 30,048 Da (residues 1–273) and a calculated isoelectric point of 5.1. QAPRTase is ubiquitous in distribution and has been isolated from mammalian liver and brain, plants, and bacteria. QAPRTase provides the de novo source of nicotinate mononucleotide (NAMN) for NAD biosynthesis in both prokaryotes and eukaryotes.1, 2 QAPRTase catalyzes the formation of NAMN, carbon dioxide, and pyrophosphate from 5-phosphoribosyl 1-pyrophosphate (PRPP) and pyridine 2,3-dicarbolic acid (quinolic acid) in the presence of Mg2+. Major interest in the human central nervous system form of QAPRTase has been fueled by the observation of high quinolic acid levels in some degenerative brain diseases.2-5 Here, we report the crystal structure of TM1645 determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics.6 The structure of TM1645 [Fig. 1(A)] was determined to 2.50 Å resolution by the molecular replacement (MR) method using a search model constructed from quinolic acid phosphoribosyltransferase (QAPRTase) from Mycobacterium tuberculosis, with a sequence identity of 30% (PDB: 1QPN).5 Data collection, model, and refinement statistics are summarized in Table I. The final model includes two protein molecules (residues 1–144 and 153–273 for chain A, residues 1–144 and 152–273 for chain B) and 183 water molecules. No electron density was observed for residues 145–152 in chain A and residues 145–151 in chain B. The Matthews' coefficient (Vm) for TM1645 is 3.57 Å3/Da and the estimated solvent content is 65.2%. The Ramachandran plot, produced by PROCHECK 3.47 shows that 92.9% of the residues are in the most favored regions, 6.9% in additional allowed regions, and 0.2% in generously allowed regions. Crystal structure of TM1645. (A) Stereo Ribbon diagram of Thermotoga maritima TM1645 color coded from N-terminus (blue) to C-terminus (red) showing the domain organization and the location of the active site (arrow). Helices (H1–H11) and β-strands (β1–β12) are indicated. Figure 1A produced with PYMOL (DeLano Scientific LLC). (B) Diagram showing the secondary structure elements in TM1645 superimposed on its primary sequence. The disordered region (residues 145–152) is shown in a dashed line and the three β-sheets (A,B,C) are labeled in red. Figure 1B produced with PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum). The TM1645 monomer is composed of 12 β-strands (β1–β12) and 11 helices (H1–H11), where H2, H10, and H11 are 310-helices [Fig. 1(A,B)]. The total β-strand, α-helical and 310-helical content is 21.9, 39.6 and 3.4%, respectively. TM1645 contains an N-terminal domain (residues 1–116, 258–273) and a C-terminal domain [residues 117–257; Fig. 1(A)]. According to the Structural Classification of Proteins database (SCOP),8 the N-terminal domain classifies as an “α/β-Hammerhead” fold and the C-terminal domain as an open “TIM β/α-barrel” fold. The N-terminal α/β-domain contains four α-helices (H1–H4) and two antiparallel β-sheets, comprised of sheet A β-strands β1, β3, β5, β12, with 4132 topology, and sheet B β2 and β4. The C-terminal domain contains a six-stranded, open β/α barrel, with an almost circular cross section. The parallel β-sheet (β6–β11) has 234561 topology and is surrounded by seven helices [H5–H11; Fig. 1(A,B)]. TM1645 forms a dimer through a head-tail interaction, with a buried surface area of 2016 Å2 per monomer [Fig. 2(A)]. Studies with QAPRTase from M. tuberculosis and Salmonella typhimurium have shown that the active enzyme exists as a dimer in solution [2, 4]. (A) Ribbon diagram of the TM1645 dimer. (B) Ribbon diagram of a superposition of TM1645 (rainbow) with its structural homologue QAPRTase from Mycobacterium tuberculosis (PDB: 1QPN; white). For reference, the nicotinate mononucleotide (NAMN) from 1QPN is shown in cpk mode. (C) Superposition of the active sites in TM1645 (cyan) and 1QPN (white). The active site residues and the NAMN molecule are shown in ball and stick. Residue names (if different) and numbers for 1QPN are shown in brackets. The atoms are indicated as follows: carbon (TM1645: cyan; 1QPN: white), nitrogen (blue), oxygen (red), phosphor (purple), and sulfur (orange). Figures produced with PYMOL (Delano Scientific LLC). A structural similarity search, performed with the coordinates of TM1645 using the DALI server,9 indicates that the closest structural homologue is QAPRTase from M. tuberculosis (PDB: 1QPN) [5]. The RMSD between TM1645 and QAPRTase from M. tuberculosis is 2.4 Å over 261 aligned residues with 30% sequence identity [Fig. 2(B)]. The main differences between the two structures are located in the loop regions next to the active site (residues 123–125 and 145–152) and are most likely the result of conformational changes due to the bound NAMN molecule in QAPRTase from M. tuberculosis. The loop region between H5 and β7 (residues 145–152) is disordered in TM1645, but visible in the structure of QAPRTase where His161 forms a hydrogen bond to the carboxylate of the NAMN molecule. All active site residues essential for catalysis are conserved, which includes His147 and R148 in the disordered region [His161 and R162 in QAPRTase from M. tuberculosis; Fig. 2(C)]. According to FFAS,10 TM1645 has four distant homologues in the T. maritima proteome: TM0474 with 17% sequence identity, TM0140 (trpC) with 8% sequence identity, TM0066 with 13% sequence identity, and TM1718 with 13% sequence identity. The sequence similarity for TM0066 and TM1718 corresponds to the TM1645 C-terminal domain only. Models for TM1645 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM1645. The structure reported here represents the first thermophilic QAPRTase, whose structure has been determined by X-ray crystallography. An analysis of the native crystal structure, in conjunction with sequence alignments and studies of co-crystals of QAPRTase from Mycobacterium tuberculosis with NAMN5, reveals conservation of the active site. We expect that the information reported here will yield valuable insights into the determinants for catalysis, substrate specificity, and thermal stability of this protein family. Protein production and crystallization: TM1645 (NadC; Swissprot: Q9X1X8) was amplified by PCR from T. maritima strain MSB8 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of TM1645. The PCR product was cloned into plasmid pMH1, which encodes expression and purification tags consisting of the amino acids MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a modified Terrific Broth [24 g/L yeast extract, 12 g/L tryptone, 1% (v/v) glycerol, 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS), pH 7.6] using the Escherichia coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 1 mg/mL. Bacteria were lysed by sonication after a freeze-thaw procedure in lysis buffer [50 mM Tris, pH 7.9, 50 mM NaCl, 1 mM MgCl2, 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 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 wash buffer [50 mM potassium phosphate, pH 7.8, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP], and the protein 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) previously equilibrated with the same buffer. Protein was eluted using a linear gradient of 50 to 500 mM NaCl in Buffer Q, and appropriate fractions were pooled. Protein was 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 to ∼10 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method11 with standard JCSG crystallization protocols.6 The crystallization solution contained 10% PEG6000 and 0.1 M MES at pH 6.0. The crystals were indexed in the orthorhombic space group I222 (Table I). Native diffraction data were collected at Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, CA) on beamline 9-1 using the BLU-ICE12 data collection environment (Table I). The data set was collected at 100K using a Quantum 315 CCD detector. Data were integrated and reduced using Mosflm13 and then scaled with the program SCALA from the CCP4 suite.14 Data statistics are summarized in Table I. The structure was determined by molecular replacement using program MOLREP from the CCP4 suite.14 A homology model based on the FFAS8 alignment between TM1645 and QAPRTase from M. tuberculosis, with a sequence identity of 30% (PDB: 1QPN)5 was constructed with the modeling program Whatif15 and used as a search model. Structure refinement was performed using REFMAC5,14 program O16 and Xfit.17 Refinement statistics are summarized in Table I. The final model includes two protein molecules (residues 1–144 and 153–273 for chain A, residues 1–144 and 152–273 for chain B) and 183 water molecules in the asymmetric unit. No electron density was observed for residues 145–152 in chain A, residues 145–151 in chain B, or for the purification tag. 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 TM1645 have been deposited with the PDB and are accessible under the code 1o4u. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), 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).