|Home | About | Journals | Submit | Contact Us | Français|
Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 Å resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme.
Tartronate semialdehyde reductases (TSRs) catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis (Fig. 1a) . The enzymes are widely distributed in Eu-bacteria and have some sequence homologs in Archaea and Eukaryota . They belong to COG2084 (Clusters of Orthologous Groups, http://www.ncbi.nlm.nih.gov/COG/) currently consisting of 79 proteins which are described as 3-hydroxyisobutyrate dehydrogenase and related β-hydroxyacid dehydrogenases. TSRs belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways (for review, see ). Sequence and mutational analysis identified four distinct functional sequence motifs . The most conserved is the N-terminal cofactor binding motif consisting of highly conserved glycine and several hydrophobic residues. This motif was predicted to specifically bind NAD cofactor. The substrate-binding motif is the most distinctive feature of β-hydroxyacid dehydrogenases. A highly conserved catalytic motif consists several glycines and catalytic lysine which was found essential for enzyme activity . The C-terminal cofactor binding motif is strongly conserved in bacteria but only 2 residues, lysine and glycine, are conserved in all β-hydroxyacid dehydrogenases family members.
Here, we report the crystal structure of GarR a TSR from Salmonella typhimurium. The structure has been determined by the single-wavelength anomalous diffraction (SAD) method and refined to 1.65 Å resolution. The selenomethionine (SeMet) derivative of TSR crystallized in the I222 space group with unit cell dimensions of a = 55.28 Å, b = 105.43 Å, c = 155.04 Å, α = β = γ = 90.00°. The active site of the enzyme includes L-tartrate (Fig. 1b) which was present in crystallization solution.
The open reading frame of garR gene, coding TSR protein, was amplified by PCR from genomic S. typhimurium LT2 DNA. The gene was cloned using the ligation independent cloning method into the pMCSG7 cloning vector  containing a TEV protease cleavage site. The fusion protein was overexpressed in E. coli BL21-Gold (DE3) (Stratagene) harboring an extra plasmid encoding three rare tRNAs (AGG and AGA for Arg, ATA for Ile). The cells were grown in minimal medium M9 at 37°C to an OD600 of ~0.8 and protein expression induced with 0.1 mM IPTG in the presence of six inhibitory amino acids and SeMet . After induction, the cells were incubated overnight with shaking at 20°C. The harvested cells were re-suspended in binding buffer (500 mM NaCl, 5% glycerol, 50 mM HEPES pH 8.0, 10 mM imidazole, 10 mM 2-mercaptoethanol), flash-frozen in liquid N2 and stored at −70°C. Thawed cells were lysed by sonication after the addition of protease inhibitor cocktail (Sigma). The lysate was clarified by centrifugation (30 min at 27000g) and applied to a metal chelate affinity-column charged with Ni2+. The hexa-histidine fusion protein was eluted from the column in elution buffer (500 mM NaCl, 5% glycerol, 50 mM HEPES pH 8.0, 250 mM imidazole, 10 mM 2-mercaptoethanol) and the tag then cleaved from the protein by treatment with recombinant His-tagged TEV protease. The cleaved protein was then resolved from the cleaved His-tag and the His-tagged protease by flowing the mixture through a second Ni2+-column. The TSR protein was dialyzed in 10 mM HEPES pH 8.0, 200 mM NaCl, and concentrated using a BioMax concentrator (Millipore).
The protein was crystallized by vapor diffusion in 96-well sitting-drop plates by mixing 0.5 μl of the protein solution (107 mg/ml) with 0.5 μl of 0.1 M immidazole buffer pH 8.0, 1 M potassium/sodium L-tartrate and 0.2 M sodium chloride, and equilibrated at 16°C over 135 μl of this solution. Crystals, which appeared overnight, were flash-frozen in liquid nitrogen with crystallization buffer plus 20% glycerol as cryoprotectant prior to data collection.
Diffraction data were collected at 100 K at the 19BM beamline of the Structural Biology Center at the Advanced Photon Source, Argonne National Laboratory. The single-wavelength inverse-beam SAD data set (at peak wavelength, 0.97957 Å) was collected from a SeMet-labeled protein crystal using SBC3 CCD detector. The crystal diffracted up to 1.65 Å resolution and belonged to I222 space group with a cell dimension of a = 55.28 Å, b = 105.43 Å, c = 155.04 Å, and α = β = γ = 90°. Because of size and square detector geometry the collected data are complete only to 1.9 Å (edge of the detector). However, high signal-to-noise data were recorded at detector corners (although not complete). Therefore we included all collected data in data processing and refine-ment. All data were processed and scaled with HKL2000  to an R-merge of 10.4% (Tables 1, ,22).
Heavy atom sites were found by SHELXD program  and structure was phased using SOLVE . The initial model was built by RESOLVE program  and then model was extended with the wARP program . After one round of refinement with CNS suite  model was manually adjusted using the program O . The refinement of the structure was completed using the REFMAC 5  of the CCP4 program suite . The final model was analyzed and validated with PROCHECK  and WHATCHECK . Structure similarity analysis was performed using DALI server .
Atomic coordinates and structure factors have been deposited into the Protein Data Bank (PDB) as 1TEA (recently updated and replaced by 1VPD).
The overall structure of S. typhimurium TSR consists of two structural domains and four previously identified functional motifs can now be mapped to these domains (Fig. 2a). N-terminal domain (residues 3–162) is formed by a 9-strand β-sheet surrounded by nine α-helixes and resembles FAD/NAD(P)-binding Rossmann fold superfamily. This domain includes N-terminal cofactor binding motif (residues 6–25) and substrate binding motif (residues 119–132). The C-terminal domain (residues 164–296) is formed exclusively by seven α-helixes with one of them (helix 166–199) extruding outside the main body of the domain and reaching toward the N-terminal domain with a possible hinge at Gly163. This domain contains a catalytic motif (residues 163–176) and the C-terminal part of the cofactor binding motif (residues 240–247). The L-tartrate molecule was found bound between two structural domains. It interacts with Ser123-Gly124-Gly125, a part of the substrate-binding domain, and Gln176 and Lys172, part of the catalysis domain (Fig. 2b). There is also indirect van der Waals contact through water molecule with Met13 from the N-terminal cofactor-binding motif, and water mediated hydrogen bonds with Ser 97 and Asp 241 from C-terminal cofactor-binding motif. The orientation of the L-tartrate molecule suggests that C4 carboxylate of L-tartrate in our structure provides the majority of contacts with the protein.
Structure similarity search using the program DALI  showed that the S. typhimurium TSR structure closely resembles two other structures in Protein Data Bank (PDB): tartronic semialdehyde reductase from S. typhimurium (PDB id: 1YB4) and hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8 (PDB id: 1J3 V). The first structural homolog is an enzyme catalyzing the same reaction in S. typhimurium as the GarR. Two enzymes share 41% sequence identity. The structural homology of two structures is very high with a Z-score of 38.3 (the strength of structural similarity in standard deviations above expected) and RMSD (of superimposed atoms) equal to 2.8 Å for all residues. Tartronic semialdehyde reductase structure contains a protein dimer in asymmetric unit which likely represents a putative biological unit of the enzyme based on PISA  results (Protein Interfaces, Surfaces and Assemblies service at European Bioinformatics Institute (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html)). Very similar dimer is formed by the GarR, two monomers are related by two fold crystallographic symmetry. Interface between two GarR molecules in the dimer has buried area of 6130 Å2 based on PISA analysis .
The second structural homolog, hydroxyisobutyrate dehydrogenase, has structural homology to our structure with a Z-score of 27.2 and RMSD equal to 1.5 Å for all residues. These two proteins share 34.7% sequence identity. The hydroxyisobutyrate dehydrogenase structure contains NADPH molecule (dihydro-nicotinamide-adenine-dinucleotide phosphate) bound to protein. The NADPH interacts with cofactor-binding domain of T. thermophilus protein. The superposition of two structures places reactive site of nicotinamide ring directly next to L-tartrate in the TSR structure (Fig. 3). Tartrate terminal oxygen O11 at carbon C1 is located only 1.37 Å from reactive carbon C2 of nicotinamide ring. The real TSR substrate is tartronate semialdehyde which is shorter by carboxyl group. The oxygen O2 at carbon C2 in our structure may mimic the real terminal oxygen of tartronate. This oxygen is only 2.64 Å apart from NADPH reactive site in superposed structures. This observation strongly suggests that L-tartrate binding in our structure serves as a good model for binding tartronate to the enzyme and proper positioning substrate for enzymatic reaction. Nevertheless, experiments with an exact substrate for TSR protein, tartronate semialdehyde, should be performed for more precise results.
We wish to thank all members of the Structural Biology Center at Argonne National Laboratory for their help in conducting experiments. This work was supported by National Institutes of Health Grant GM62414, GM074942 and by the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.
The submitted manuscript has been created by UChicago Argonne, LLC, Operator of Argonne National Laboratory (“Argonne”). Argonne, a US Department of Energy Office of Science laboratory, is operated under Contract No. DE-AC02-06CH11357. The US Government retains for itself, and others acting on its behalf, a paid-up nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the Government.
J. Osipiuk, Biosciences Division, Midwest Center for Structural Genomics, Argonne National Laboratory, 9700 South Cass Ave., Bldg 202, Argonne, IL 60439, USA.
M. Zhou, Biosciences Division, Midwest Center for Structural Genomics, Argonne National Laboratory, 9700 South Cass Ave., Bldg 202, Argonne, IL 60439, USA.
S. Moy, Biosciences Division, Midwest Center for Structural Genomics, Argonne National Laboratory, 9700 South Cass Ave., Bldg 202, Argonne, IL 60439, USA.
F. Collart, Biosciences Division, Midwest Center for Structural Genomics, Argonne National Laboratory, 9700 South Cass Ave., Bldg 202, Argonne, IL 60439, USA.
A. Joachimiak, Biosciences Division, Midwest Center for Structural Genomics, Argonne National Laboratory, 9700 South Cass Ave., Bldg 202, Argonne, IL 60439, USA. Biosciences Division, Structural Biology Center, Argonne National Laboratory, 9700 South Cass Ave., Bldg 202, Argonne, IL 60439, USA. Email: vog.lna@jjezrdna.