Crystal structure of an iron‐containing 1,3‐propanediol dehydrogenase (TM0920) from Thermotoga maritima at 1.3 Å resolution (2003)

The TM0920 gene of Thermotoga maritima encodes a predicted iron-containing 1,3-propanediol dehydrogenase (EC 1.1.1.202) with a molecular weight of 39,944 Da and a predicted isoelectric point of 6.1. 1,3-propanediol dehydrogenase catalyzes the oxidation of propane-1,3-diol to 3-hydroxypropanal with the simultaneous reduction of NADP+ to NADPH. Another four alcohol dehydrogenase paralogues of TM0920 have been identified in the Thermotoga proteome (TM0111, TM0285, TM0423, and TM0820), as well as hundreds of orthologues in the three Domains of Life. Here, we report the crystal structure of TM0920 determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics.1 The crystal structure is consistent with its predicted function. The crystal structure of TM0920 (Fig. 1A) was determined to 1.3 Å resolution using the multiple-wavelength anomalous dispersion (MAD) method. Data collection and refinement statistics are summarized in Table I. The final model includes two protein molecules (residues 1–359), two nicotinamide-adenine dinucleotide phosphate molecules (NADP+), two Fe2+ ions, and 895 water molecules. The Matthews' coefficient (Vm) for TM0920 is 2.15 Å3/Da and the estimated solvent content is 42.8%. The Ramachandran plot produced by PROCHECK 3.42 shows that 92.8% of the residues are in the most favored regions, 7.1% in additional allowed regions and 0.2% in generously allowed regions. No residues lie in disallowed regions. Crystal structure of TM0920. A: Ribbon diagram of Thermotoga maritima TM0920 1,3-propanediol dehydrogenase color coded from N-terminus (blue) to C-terminus (red) showing the domain organization and location of the active site (arrow). α-helices (H1–H19) and β-strands (β1–β8) are indicated. The NADP+ molecule is shown in ball and stick. The Fe2+ ion is shown as a sphere in magenta. B: Diagram showing the secondary structure elements in TM0920 superimposed on its primary sequence. Residues located in the active site and interacting with Fe2+ are indicated by blue dots and green triangles. Residues interacting with NADP+ are indicated by red dots. The location of the β-hairpin formed by β-strands 6 and 7 is depicted in red. The TM0920 monomer consists of a single polypeptide chain of 359 residues, composed of 19 helices (13 α-helices, and 6 310-helices), and 8 β-strands (Fig. 1B). The total α-helix, 310-helix, and β-strand content is 48.7, 7.2, and 12.3%, respectively. Each monomer has two distinct domains separated by a deep cleft. The α/β N-terminal domain A (residues 1 to 177) consists of 8 β-strands (β1 to β8) and 9 helices (H1 to H9), arranged as a 6-stranded parallel β-sheet (β1 to β5, and β8, with a topology −4X −1X 2X 1X 1X) flanked by α-helices in a Rossmann-fold topology, and two β-strands (β6 and β7) forming a β-hairpin. The C-terminal domain B (residues 178 to 359) comprises helices H10 to H19 organized in two helical bundles. The TM0920 structure represents a dehydroquinate synthase-like fold7 and is the third member determined for the glycerol dehydrogenase-like structural family (together with 1KQ3 and 1JQA). Analogy with other dehydrogenases and analysis of the crystal packing suggest that a monomer is the biologically-relevant form. A BLAST search of the TM0920 protein versus the Swissprot database indicated that the highest level of sequence identity (32% identity, 47% considering conserved and partially conserved residues) corresponded to proteins from Klebsiella pneumoniae (Swissprot: Q59477) and Citrobacter freundii (Swissprot: P44513). Accordingly, TM0920 was annotated as an iron-dependent 1,3-propanediol dehydrogenase. Biochemical data for these two proteins3, 4 were used as supporting evidence to model density in the active site as an Fe2+ ion. The Fe2+ ion is deeply located in the catalytic cleft and has a square pyramidal coordination with Asp189, His193, His256, and His270. All of these residues are situated in domain B (Fig. 2B). The NADP+ binding site is located between domains A and B. The interactions between NADP+ and the surrounding residues in the active site are depicted in Figure 2A. Interestingly, the density for the fully refined model is weak for the nicotinamide ring and shows residual difference density peaks near the nicotinamide NC5 atom. In addition, the side-chain of the metal coordinating His256 has a dual conformation and the Fe2+ ion, refined with SHELX,16 appears to be only partially (60%) occupied. These findings indicate a possible superposition of states in the active site, which could not be completely accounted for with the current model. A: Schematic representation of the interactions between NADP+ and its interacting residues. The NADP+ molecule is depicted in purple and the protein residues are in orange. The atoms are indicated as follows: carbon (black), oxygen (red), nitrogen (blue), and phosphorus (purple). Hydrogen bonds are represented as dashed green lines. Residues implicated in hydrophobic interaction are represented as barbed circle sections. B: Close-up view of the active site showing residues coordinating the metal ion. A structural similarity search, performed using the DALI server5 indicated that TM0920 is structurally similar to a glycerol dehydrogenase from Bacillus stearothermophilus (PDB: 1JQA),6 with an RMSD value of 3.0 Å for the superimposition of 321 Cα atoms with 20% sequence identity. A structural alignment between TM0920 and TM0423 (PDB:1KQ3), another alcohol dehydrogenase from Thermotoga maritima whose structure has also been determined by the Joint Center for Structural Genomics,8 has an RMSD value of 3.1 Å for the superimposition of 322 Cα atoms with 21% sequence identity. According to the Structural Classification of Proteins database (SCOP),7 the TM0920 structure has a dehydroquinate synthase-like fold, and represents the third member of the glycerol dehydrogenase-like structural family (together with 1KQ3 and 1JQA). Models for a number of TM0920 orthologues and paralogues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM0920 The TM0920 structure represents an iron-containing 1,3-propanediol dehydrogenase in complex with NADP+. We expect that the information reported here will yield valuable insights into the determinants for catalysis, substrate specificity, and thermal stability of this family of alcohol dehydrogenases. An iron-containing 1,3-propanediol dehydrogenase (TIGR: TM0920; Swissprot: Q9X022) was PCR amplified using Pfu (Stratagene) from Thermotoga maritima strain MSB8 genomic DNA using primer pairs encoding the predicted 5′- and 3′-ends of TM0920. The PCR product was cloned into plasmid pMH1, which encodes a purification tag consisting of the 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. Bacteria were lysed by sonication after a freeze-thaw procedure in Lysis Buffer (50mM Tris pH 7.9, 50mM NaCl, 1mM MgCl2, 0.25mM Tri (2-carboxyethyl) phosphine hydrochloride (TCEP), 1mg/ml lysozyme) and cell debris pelleted by centrifugation at 3600× g for 60 minutes. The soluble fraction was applied to a nickel chelate resin (Pharmacia) previously equilibrated with Equilibration Buffer (50mM KH2PO4 pH 7.8, 0.25mM TCEP, 10% v/v glycerol, 0.3M NaCl) containing 20 mM imidazole. The resin was washed with Equilibration Buffer containing 40 mM imidazole, and protein was eluted with Elution Buffer (20 mM Tris pH 7.9, 10% v/v glycerol, 0.25 mM TCEP, 300 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, 50 mM NaCl, 5% v/v glycerol, 0.25 mM TCEP) was then applied to a Resource Q column (Pharmacia). Protein was eluted using a linear gradient to 500 mM NaCl. Appropriate fractions were buffer exchanged into size exclusion chromatography (SEC) Buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP). The protein was concentrated for crystallization assays by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method9 with standard JCSG crystallization protocols.1 The crystallization buffer contained 20% PEG-300, 5% (w/v) PEG-8000, 10% glycerol, and 0.1 M Tris-HCl at pH 8.5. The crystals were indexed in the monoclinic space group P21 (Table I). Native and multi-wavelength anomalous diffraction data were collected at Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, USA) on beamline 11-1 at multiple wavelengths using the BLU-ICE10 data collection environment (Table I). All data sets were collected at 100 K using a Quantum 315 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. The structure was determined using the software packages SnB,13 the CCP4 suite,12 and SOLVE.14 An initial model was built using the ARP/wARP package.15 The structure was refined to a resolution of 1.3 Å using REFMAC512 and SHELX.16 Refinement statistics are summarized in Table I. The final model contains two protein molecules (residues 1 to 359), two NADP+ molecules, two Fe2+ ions, two TRIS (2-amino-2-hydroxymethyl-propane-1,3-diol) molecules and 895 water molecules in the asymmetric unit. The model for chain B contains two residues, His −1 and 0, from 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 TM920 have been deposited in the PDB and are accessible under the code 1O2D. This work was supported by NIH Protein Structure Initiative grant P50-GM 62411 from the National Institute of General Medical Sciences (www.nigms.nih.gov). 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). The JCSG is grateful for constructive comments from Christian Cambillau.