Crystal structure of a zinc‐containing glycerol dehydrogenase (TM0423) from Thermotoga maritima at 1.5 Å resolution (2002)

The TM0423 gene of Thermotoga maritima encodes a zinc-containing glycerol dehydrogenase (EC 1.1.1.6) with an molecular weight of 39,693 Da and a predicted isoelectric point of 5.2. Glycerol dehydrogenase catalyzes the oxidation of glycerol to dihydroxyacetone with the simultaneous reduction of oxidized nicotinamide adenine dinucleatide (NAD+) to reduced nicotinamide adenine dinucleotide (NADH). In this work, we report the crystal structure of TM0423 determined using a semiautomated high-throughput pipeline at the Joint Center for Structural Genomics.1 Crystals of TM0423 diffracted to 1.5 Å resolution, with the average I/σ(I) value being 17.2 for all reflections and 4.3 in the highest resolution shell (1.5–1.54 Å). A total of 61,417 reflections were observed. This data set is 99.4% complete. We determined the structure of TM0423 using the multiple-wavelength anomalous dispersion (MAD) technique (Table I). The final model includes residues 1–363, a Tris buffer molecule, 227 water molecules, a Zn2+ ion, and a Cl− ion. The Ramachandran plot produced by PROCHECK 3.42 shows 93.4% of all residues in the most favored regions, 6.3% in additional allowed regions, and 0.3% in generously allowed regions. No residues lie in disallowed regions. TM0423 consists of a single polypeptide chain (364 residues), composed of 18 α helices (α1–α18) and 8 β strands (β1–β8). Total α helix and β strand content is 46.1% and 11.4%, respectively (Fig. 1). The structure has two distinct domains separated by a deep cleft. The N-terminal domain A consists of the 8 β strands and 7 helices (residues 1–158), arranged as a parallel β sheet flanked by α helices. The C-terminal B domain (residues 159–364) comprises helices 8–18 organized in two helical bundles. Ribbon diagram of Thermotoga maritima TM0423 glycerol dehydrogenase showing the domain organization and location of the catalytic cleft. Strands are shown in cyan, and helices are shown in red. The Tris-buffer molecule is shown in a stick representation. The Zn2+ ion is shown in blue. A Zn2+ ion was modeled based on structural homology to glycerol dehydrogenase from Bacillus stearothermophilus. The Zn2+ ion is located deep in the catalytic cleft and is coordinated by residues His252, His269, and Asp169 (Fig. 2). The glycerol-binding site is occupied by a Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) molecule from the buffer. The Tris molecule is positioned in the catalytic site through coordination to the Zn2+ ion, where it contributes 2 of the coordinating ligands. Three B domain residues (His252, His269, Asp169) contribute another 3 coordinating ligands to the Zn2+ (Fig. 2). Phenylalanine 243 is also involved in positioning the Tris-pseudo substrate through hydrophobic interactions. Diagram of the active site region showing the relative location of Zn2+ and Tris, and the possible network of interactions contributing to the correct positioning of a polyol substrate in the active site of the enzyme. A structural similarity search, performed with the DALI server,3 with the coordinates of TM0423, indicated that this protein is structurally similar to a mutant Bacillus stearothermophilus glycerol dehydrogenase [Protein Data Bank (PDB) accession code 1JPU],4, 5 with root-mean-square difference (RMSD) values of 1.6 Å for the superimposition of 357 Cα atoms. Sequence alignment of the superimposed structures showed 48% sequence identity. According to Structural Classification of Proteins (SCOP) database,6 these two glycerol dehydrogenases are the only members of the glycerol dehydrogenase family with a dehydroquinate synthase–like fold. This fold contains two domains. Domain 1 is an α/β domain with Rossman-fold topology, capable of binding NAD+, and Domain 2 is a multihelical array. B. stearothermophilus is a thermophilic bacterium whose optimal growth temperature is 55°C. In contrast, T. maritima is a hyperthermophilic bacterium whose optimal growth temperature is 80°C, but can grow in temperatures approaching the boiling point of water. Thus, the differences observed between these two structures and sequences of both glycerol dehydrogenases may yield valuable insights into the determinants for thermal stability in this protein family. Protein production: Glycerol dehydrogenase (TIGR: TM0423; GenBank: AAD35508) was PCR amplified using Pfu (Stratagene) from T. maritima strain MSB8 genomic DNA, with primer pairs encoding the predicted 5′ and 3′-ends of TM0423. The PCR product was cloned into plasmid pMH1, which encodes a 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 with the use of the Escherichia coli methionine auxotrophic strain DL41. 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, 3 mM DL-methionine, 0.25 mM Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), 1 mg/mL lysozyme) and cell debris pelleted by centrifugation at 3600× g for 60 min. The soluble fraction was applied to a nickel chelate resin (Invitrogen) previously equilibrated with equilibration buffer (20 mM Tris pH 7.9, 0.25 mM TCEP, 10% v/v glycerol, 3 mM DL-methionine). The resin was washed with equilibration buffer containing 40 mM imidazole and protein eluted with equilibration buffer containing 200 mM imidazole. Buffer exchange was performed to remove imidazole from the protein eluate, and the protein in buffer Q (20 mM Tris pH 7.9, 25 mM NaCl, 5% v/v glycerol, 0.25 mM TCEP) was applied to a Resource Q column (Pharmacia). Protein was eluted using a linear gradient to 400 mM NaCl. We further purified appropriate fractions by size-exclusion chromatography (SEC) using S200 resin (Pharmacia) with isocratic elution in SEC buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP). The protein was concentrated to 7.8 mg/mL by centrifugal ultrafiltration (Millipore). Crystallization: The protein was initially crystallized using the vapor diffusion method with 50 nl of protein and 50 nl of mother liquor in sitting drops on customized microtiter plates (Greiner). Each protein was set up with 480 standard crystallization conditions [Wizard I/II. Cryo I/II (Emerald BioStructures, Bainbridge Island, WA), Crystal Screen, Crystal Screen 2, Crystal Screen Cryo, PEG/Ion Screen, Grid Screen Ammonium Sulfate, Grid Screen PEG 6000, Grid Screen MPD, and Grid Screen PEG/LiCL (Hampton Research, Riverside, CA)] at 20°C. Images of each crystal trial were taken at 0, 7, and 28 days after setup with an Optimag Vecco Oasis 1700 imager. Images at days 7 and 28 were also evaluated manually. To produce the final crystals used for data collection, 1 μl drops were used. The crystallization buffer contained 35% 2-Methyl-2,4-pentane diol (MPD) as the precipitant in 0.1 M Na/K phosphate at pH 6.2. Crystals grew within 28 days at 20°C. The crystals indexed in the tetragonal space group I422 (Table I). Data collection: Native and multiple wavelengths MAD data were collected at SSRL (Stanford, USA) on beamline 9-2 using the BLU-ICE data-collection program (Table I). All data sets were collected at 100°K with a Quantum 4 charge-coupled device (CCD) detector. Data were reduced using Mosflm7 and then scaled with the program SCALA from the CCP4 suite.8 Data statistics are summarized in Table I. Structure solution and refinement: The structure was determined with use of the software packages SnB,9 the CCP4 suite8 program MLphare, and SOLVE.10 We built an initial model using the ARP/wARP package.11 The structure refinement was performed using CNS.12 Refinement statistics are summarized in Table I. The final model contains residues 1 to 363. The C-terminal threonine, as well as the purification tag, were excluded from the model as no electron density was found for these residues. Validation and deposition: Analysis of the stereochemical quality of the models was accomplished with use of 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 TM0423 have been deposited with the PDB and are accessible under the code 1KQ3.