Crystal structure of a putative oxalate decarboxylase (TM1287) from <i>Thermotoga maritima</i> at 1.95 Å resolution

Abstract

The TM1287 gene of Thermotoga maritima encodes a conserved hypothetical protein with a molecular weight of 13,138 Da (residues 1–121) and a calculated isoelectric point of 5.6. Currently, no functional annotation has been made for this protein. Here, we report the crystal structure of TM1287 in complex with an endogenously bound oxalate determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).1 Based on the structure of the TM1287/oxalate complex and its similarity to oxalate decarboxylase (OXDC) from Bacillus subtilis [Protein Data Bank (PDB) code: 1j58),2 we propose that TM1287 functions as a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide.3 The structure of TM1287 [Fig. 1(A)] was determined to 1.95 Å resolution using the molecular replacement (MR) method, with a search model constructed from Auxin-binding protein (PDB code: 1LR5), despite an extremely low sequence identity (∼15%). Data collection, model, and refinement statistics are summarized in Table I. The final model includes 2 protein molecules (residues 7–121), 2 manganese ions, 2 endogenous oxalate molecules, and 251 water molecules. No electron density was observed for residues 1–6. The Matthews coefficient (Vm) for TM1287 is 2.41 Å3/Da, and the estimated solvent content is 47.0%. The Ramachandran plot produced by PROCHECK 3.44 shows that 95% of the residues are in the most favored regions, and 5% are in additional allowed regions. Crystal structure of TM1287. (A) Ribbon diagram of Thermotoga maritima TM1287 color coded from N-terminus (blue) to C-terminus (red), showing the domain organization and the location of the manganese ion (purple sphere). The 310-helix H1, β-strands (β1–β11) and β-sheets A and B are indicated. (B) Ribbon diagram of the TM1287 dimer. β-strands β1 and β9 of the crossover interaction are indicated. Figures produced with PYMOL (DeLano Scientific LLC). The final model of the TM1287 monomer consists of a single polypeptide chain of 115 amino acids composed of 11 β-strands (β1–β11) and 1 short 310-helix (H1). The total β-strand content is 43.6%. The TM1287 structure is characterized by two antiparallel β-sheets (A and B) that form a jelly roll β-sandwich with a topology that is characteristic of the cupin barrel fold3, 5 [Fig. 1(A)]. The 7-stranded β-sheet A has a 2 3 4 11 6 9 1 topology, where β-strand β1 is contributed from the neighbor subunit in the dimer. The 4-stranded β-sheet B has 5 10 7 8 topology [Fig. 2(A)]. Each of the 11 β-strands is approximately perpendicular to the barrel axis. TM1287 forms a dimer linked by a pair of crossovers between the adjacent edge β-strands, β1 and β9, from different subunits in the dimer [Fig. 1(B)]. The dimer interface corresponds to interactions between the two A β-sheets with a buried surface area of 775 Å2 per monomer. (A) Diagram showing the secondary structure elements in TM1287 superimposed on its primary sequence. β-hairpins are depicted in red. Residues coordinating the metal ion and the oxalate ligand are marked with blue and red dots, respectively. Figure 2A from PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum). (B) The active site of TM1287 is depicted showing the manganese ion (Mn), its coordinating residues (His61, His63, Glu68, His102), and a bound oxalate molecule (OXL) in ball and stick configuration. The atoms are indicated as follows: Carbon (gray), oxygen (red), nitrogen (blue), and manganese (purple). Hydrogen bonds are represented as dashed yellow lines. Figure 2B produced with PYMOL (DeLano Scientific LLC). Each TM1287 domain has a metal-binding site in the mouth of the β-barrel [Fig. 2(B)]. The metal ion has octahedral coordination, in which 4 ligands are contributed by histidine and glutamate side-chains that are highly conserved in the cupin family.3 The metal-binding residues are His61, His63, Glu68, and His102, with metal-to-atom distances of 2.13, 2.08, 2.18, and 2.23 Å, respectively. The remaining coordination sites are occupied by 2 carboxyl oxygen atoms of an oxalate molecule at a distance of 2.19 and 2.21 Å, respectively [Fig. 2(B)]. The oxalate molecule probably originated from the bacterial expression system, because it was not added to the purification or crystallization buffers. According to the octahedral coordination sphere and its structural similarity to OXDC,2 the metal appears to be manganese. Difference density continuous with the guanidine group of Arg23, which is adjacent to the oxalate, suggests a ω-hydroxylated catalytic arginine, which would be consistent with the proposed catalytic mechanism.2 Since the density suggests only partial occupancy for this species, it has been modeled as a closely bound water molecule. A structural similarity search, performed with the coordinates of TM1287 using the DALI server,6 indicates that the closest structural homologue is OXDC (PDB code: 1J58).2 The root-mean-square deviation (RMSD) between TM1287 and OXDC is 1.2Å over 98 aligned residues with 23% sequence identity. Another structural homologue is the manganese-dependent oxalate oxidase (germin) from Hordeum vulgare (PDB code: 1fi2).7 The RMSD between TM1287 and germin is 2.5 Å over 105 aligned residues with 16% sequence identity. According to Fold and Function Assignment System (FFAS),8 TM1287 has at least five distant homologues in the T. maritima proteome: TM0656 with 20% sequence identity, TM1459 with 18% sequence identity, TM1010 with 13% sequence identity, and TM1112 with 14% sequence identity. Models for TM1287 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=tm1287. Based on the structural homology of TM1287 to OXDC from B. subtilis and the bound oxalate molecule in the active site, we propose that TM1287 functions as an OXDC in T. maritima. The structure reported here represents a putative OXDC in complex with oxalate, whose structure has been determined by X-ray crystallography. We expect that the information reported here will yield valuable insights into the determinants for catalysis and substrate specificity of this protein family. Protein production and crystallization: TM1287 (TIGR: TM1287; Swissprot: Q9X113) 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 TM1287. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag 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 Equilibration Buffer containing 40 mM imidazole, and the protein eluted with Elution Buffer [20 mM Tris pH 7.9, 10% (v/v) glycerol, 0.25 mM TCEP, 300 mM imidazole]. The nickel affinity eluate was buffer exchanged into size exclusion chromatography SEC Buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP) and concentrated to ∼10mg/mL for crystallization assays by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method,9 with standard JCSG crystallization protocols.1 The crystallization solution contained 20% polyethylene glycol (PEG) 6000, 1.0 M LiCl, and 0.1 M 2(N-morpholino) ethanesulfonic acid (MES) pH 6.0. The crystals were indexed in the monoclinic space group C2 (Table I). Data collection: Native diffraction data were collected at Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, CA) on beamline 9-1 using the BLU-ICE10 data collection environment (Table I). The data set was collected at 100 K using a Quantum 315 charge-coupled device (CCD) detector. Data were integrated and reduced using Mosflm11 and then scaled with the program SCALA from the CCP4 suite.12 Data statistics are summarized in Table I. Structure solution and refinement: The structure was determined by MR using the program MOLREP from the CCP4 suite.12 A homology model based on the FFAS8 alignment between TM1287 and the Auxin-binding protein (PDB code:1LR5), with a sequence identity of only 15% was constructed with the modeling program WHATIF13 and used as a search model. Structure refinement was performed using REFMAC5,12 O,14 and Xfit.15 Refinement statistics are summarized in Table I. The final model includes 2 protein molecules (residues 7–121), 2 manganese ions, 2 oxalate molecules (copurified), and 251 water molecules in the asymmetric unit. No electron density was observed for residues 1–6 and the His-tag. Validation and deposition: Analysis of the stereochemical quality of the models was accomplished using the JCSG Validation Central suite, which integrates 7 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 TM1287 have been deposited with the PDB and are accessible under the code 1o4t. 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).