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4.3.3.7: 4-hydroxy-tetrahydrodipicolinate synthase

This is an abbreviated version!
For detailed information about 4-hydroxy-tetrahydrodipicolinate synthase, go to the full flat file.

Word Map on EC 4.3.3.7

Reaction

pyruvate
+
L-aspartate-4-semialdehyde
=
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate
+
H2O

Synonyms

Aq_1143, AT2G45440, BA3935 gene product, cDHDPS, CjDHDPS, DapA, DapA2, DHDPA synthase, DHDPS, DHDPS2, dihydro-dipicolinic acid synthase, dihydrodipicolinate synthase, dihydrodipicolinic acid synthase, dihydrodipocolinate synthase, dihydropicolinate synthetase, EC 4.2.1.52, FaDHDPS, HTPA synthase, More, MosA, MosA protein, MRSA-DHDPS, PA1010, pyruvate-aspartic semialdehyde condensing enzyme, Rv2753c, synthase, dihydrodipicolinate, VEG81, Vegetative protein 81

ECTree

     4 Lyases
         4.3 Carbon-nitrogen lyases
             4.3.3 Amine-lyases
                4.3.3.7 4-hydroxy-tetrahydrodipicolinate synthase

Crystallization

Crystallization on EC 4.3.3.7 - 4-hydroxy-tetrahydrodipicolinate synthase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure analysis, PDB ID 2HMC, structure comparisons
crystal structure analysis, PDB ID 2R8Wm, structure comparisons
crystal structure analysis, PDB ID 3B4U, structure comparisons
crystal structure analysis, PDB IDs 4I7U, 4I7V, and 4I7W, resolution at 1.42-1.69 A, structure comparisons
purified recombinant His-tagged enzyme in the unliganded form and in forms with bound substrate and with bound substrate plus allosteric inhibitor lysine, hanging drop vapour diffusion method, mixing of 0.002 ml of 10 mg/ml protein in 20 mM Tris, pH 8.0, with 0.002 ml of reservoir solution containing 0.1 M Tris, pH 8.5, and 2 M ammonium sulfate, for ligand-bound enzyme from 0.17 M lithium sulfate monohydrate, 0.085 M Tris pH 8.5, 25.5%(w/v) PEG 4000, 15%(v/v) glycerol with addition of 20 mM pyruvate or 20 mM pyruvate and 20 mM lysine, equilibration against 1 ml reservoir solution, 20°C, overnight, X-ray diffraction structure determination and analysis at 1.40-1.55 A resolution
purified recombinant enzyme, sitting drop vapour diffusion method, mixing of 0.001 ml of 21.8 mg/ml protein in 20 mM Tris-HCl at pH 8.0 and 0.2 M NaCl, with 0.001 ml of reservoir solution containing 4 M sodium phosphate, 0.1 M imidazole, and 0.2 M NaCl at pH 8.0, 18°C, 1 week, X-ray diffraction structure determination and analysis at 1.90 A resolution, molecular replacement
purified recombinant His-tagged enzyme, sitting drop vapour diffusion method, 0.0015 ml of 14.5 mg/ml protein in 20 mM Tris-HCl, pH 8.0 is mixed with 0.0015 ml of reservoir solution containing 20% w/v PEG 6000, 200 mM sodium chloride, 100 mM Tris-HCl, pH 8.0, including 0.02% w/v sodium azide, 20°C, method screening, 5 days to 8 weeks, X-ray diffraction structure determination and analysis at 2.5 A resolution
in complex with pyruvate, by sitting- and hanging-drop vapor diffusion method, to 2.15 A resolution, shares the same space group, unit cell parameters, and a similar resolution to the structure of substrate unbound DHDPS. Twelve more hydrogen bond interactions at both interfaces in the crystal structure of pyruvate-bound DHDPS relative to the apo structure
Q81WN7
in the presence of pyruvate, the hanging-drop vapour-diffusion method, at a resolution of 2.15 A. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.5, b = 124.6, c = 131.0 A, beta = 90.0
Q81WN7
sitting-drop vapor-diffusion and hanging-drop vapor diffusion
Q81WN7
sitting-drop vapor-diffusion method at room temperature
sitting-drop vapor-diffusion
sitting-drop vapor-diffusion, protein crystals are grown in conditions consisting of 20%(w/v) PEG 4000, 100 mM sodium citrate tribasic pH 5.5 and diffract to 2.10 A resolution. They belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 79.96, b = 106.33, c = 136.25 A
crystals are grown in a solution of 0.28 M sodium acetate, 30% PEG 4000, 0.1 M TRIS pH 8.5 using the hanging drop vapor diffusion method
-
crystallized in a number of forms, predominantly using PEG precipitants, estimated solvent content of 41%, best crystal diffracting to beyond 1.9 A resolution
-
in the presence of its substrate pyruvate, by the sitting-drop vapour diffusion method, to 1.2 A resolution. Belongs to space group C2, in contrast to the unbound form, which has trigonal symmetry. Unit-cell parameters are a = 143.4, b = 54.8, c = 94.3 A , beta = 126.3. The crystal volume per protein weight is 2.3 A3 Da-1 (based on the presence of two monomers in the asymmetric unit), with an estimated solvent content of 46%
-
hanging drop method, data collection and refinement statistics, refinement resolution originally extended to 3.0 A, gradually increased to 2.5 A, and finally to 2.2 A, structural similarities to other dihydrodipicolinate synthase
by the hanging drop-vapour diffusion method, mutants K161A and K161R solved at resolutions of 2.0 and 2.1 A, respectively. They show no changes in their secondary or tertiary structures when compared to the wild-type structure. Crystal structure of mutant K161A with pyruvate bound at the active site solved at a resolution of 2.3 A, reveals a defined binding pocket for pyruvate that is thus not dependent upon lysine 161
complexed with pyruvate, the substrate analogs succinate alpha-semialdehyde and alpa-ketopimelic acid, the inhibitor dipicolinic acid, and the natural feedback inhibitor L-lysine, hanging drop vapor diffusion method, in the presence of beta-octyl glucoside using either potassium phosphate buffer (pH 10) or potassium citrate buffer (pH 7.0) as the precipitant
crystal structure at 2.5 A resolution
-
crystal structure of mutant enzyme R138H and R138A, hanging-drop vapor diffusion method
examination of the specificity of the active site of DHDPS, co-crystallization with the substrate analogue oxaloacetate, data-collection and refinement statistics
hanging-drop vapour-diffusion method, crystal struture of native and (S)-lysine-bound dihydropicolinate synthase are presented to 1.9 A and 2.0 A resolution, respectively
in complex with beta-hydroxypyruvate, hanging drop vapor-diffusion method, data processing and refinement statistics
mutant T44S, crystals are isomorphous to those of the wild-type enzyme, no significant modification in its tertiary or quaternary structure from that of the wild-type enzyme
mutant Y107W, hanging-drop vapor diffusion method, diffraction to beyond 2.0 A resolution, data collection and refinement statistics, solid-state structure of the mutant enzyme largely unchanged
purified enzyme in complex with pyruvate and substrate analogue succinic acid semialdehyde, hanging drop vapor diffusion method, mixing of 0.003 ml of 8 mg/mL protein in 20 mM Tris-HCl, pH 8.0, with 0.0012 ml of precipitant solution containing 1.8 M K2HPO4, pH 10, and 0.0006 ml of N-octyl-beta-R-glucopyranoside 6% w/v, 4°C, 3-4 days, soaking in cryoprotectant solution containing 1.8 M K2HPO4, pH 10, glycerol 20% v/v, and 120 mM succinic acid semialdehyde and 40 mM pyruvate, X-ray diffraction structure determination and analysis at 2.3 A resolution
purified recombinant wild-type and mutant enzymes, hanging or sitting drop vapour diffusion method, at 4°C and 21°C, 0.006 ml protein solutions: about 10 mg/ml protein, 1.8 M K2PO4, pH 10.0, N-octyl-beta-R-glucopyranoside 6% w/v, + 2 ml reservoir solution: 1.8 M K2PO4, pH 10.0, 3-4 days, X-ray diffraction structure determination and analysis at 2.35-2.5 A resolution
at 239K using the hanging drop-vapor diffusion method, at 1.5 A resolution. The four subunits of the asymmetric unit assemble to form a tetramer with an approximate 222 symmetry. At the active site, three residues Tyr132, Thr43 and Tyr106 are observed to constitute a catalytic triad. Has a unique extensive dimer–dimer interface that is mediated by strong hydrophobic interactions supplemented by two sets of three hydrogen bonds between four polar residues. Belongs to space group P2(1)2(1)2(1) with cell parameters a = 67.03 A, b = 120.52 A, c = 161.1 A
purified recombinant enzyme, hanging drop vapour diffusion method, mixing of 0.002 ml of 11 mg/ml protein in 20 mM Tris, pH 7.5, with 0.002 ml of reservoir solution containing 0.2 M magnesium chloride, 10% w/v PEG 8000, 0.1 M Tris chloride, pH 10, at 8°C, X-ray diffraction structure determination and analysis at 1.65-1.74 A resolution, molecular replacement
-
by the oil-batch method at 291 K, to 2.2 A resolution. Belongs to space group P21 with unit-cell parameters a = 80.5 A, b = 76.5 A, c = 101.9 A, gamma = 106.9 A. The asymmetric unit contains four DHDPS molecules, forming a homotetramer with approximate 222 symmetry. The overall tertiary structure of DHDPS possesses a (beta/alpha)8-barrel fold (TIM barrel) with three additional alpha-helices (alpha9-alpha11) at the C-terminus of the chain. The beta-strands of the barrel form an intrinsic network of hydrogen-bonding interactions with the neighbouring beta-strands and are oriented in the same directions. The functional residue Lys161, which participates in Schiff-base formation, is located within the beta-barrel and the side chain of Tyr132 sits over this residue
hanging-drop vapour-diffusion method, crystallizes in a monoclinic crystal form
-
micro-batch method and hanging drop vapour diffusion methods of crystallization
purified recombinant DHDPS mutant A204R, sitting drop vapour diffusion method at room temperature, 200 nl of 10 mg/ml protein in 20 mM Tris-HCl, 2 mM 2-mercaptoethanol, 250 mM NaCl, 5% v/v glycerol, 10 mM pyruvate, pH 8.0, is mixed with 230 nl reservoir solution containing 2.0 M ammonium sulphate, 100 mM sodium acetate, pH 5.5, X-ray diffraction structure determination and analysis at 2.0 A resolution
-
three-dimensional structure is determined and refined at 2.28 A resolution
to 2.0 A resolution, space group of the crystal is P212121, with unit cell dimensions of a = 80.7, b = 115.7 and c = 132.1 A. The secondary and tertiary structures are remarkably similar to that of Escherichia coli DHDPS. The hydrogen bond lengths within the catalytic triad, and particularly that between Y133 and T44, differ significantly from those of the Escherichia coli enzyme
crystal structure at 2.8 A resolution
-
purified recombinant DHDPS, free or in complex with inhibitor (S)-lysine, 15 mg/ml protein in 50 mM Tris-HCl, pH 8.5, at 20°C using hanging drop vapour diffusion method, mixing of 0.005 ml protein solution with 0.005 ml well solution containing 30% w/v PEG-3350, 170 mM MgCl2, 70 mM Tris-HCl, pH 8.5, and 6% v/v propylene glycol. Crystals obtained without 6% propylene glycol are soaked in the reservoir containing 20 mg/ml (S)-lysine, X-ray diffraction structure determination and analysis at 2.65-2.85 A resolution
purified recombinant His6-tagged enzyme, hanging drop vapour diffusion method, mixing of 0.002 m of 12.5 mg/ml protein solution with 0.002 ml of reservoir solution containing 18% of PEG6000, 0.2 M MgCl2, and 0.1 M TRIS-HCl, pH 7.6, X-ray diffraction structure determination and analysis at 1.6 A resolution, molecular replacement
purified recombinant His-tagged enzyme, hanging-drop vapour-diffusion method, 7.6 mg/ml protein in 150 nl solution is mixed with 150 nl of reservoir solution containing 200 mM ammonium sulfate, 100 mM Bis-Tris pH 5.0-6.0, 23-26% w/v PEG 3350, 0.02% w/v sodium azide, X-ray diffraction structure determination and analysis at 2.5 A resolution
-
crystallization conditions are optimized using vapour diffusion in hanging drops at room temperature. Crystal grown in the presence of pyruvate diffracted X-rays to 2.3 A resolution using synchrotron radiation and belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 69.14, b = 138.87, c = 124.13 A
-
structure of MosA protein solved to 1.95 A resolution, data collection and refinement statistics
atomic resolution at 1.45 A, crystal structure confirms the dimeric quarternary structure, reveals that the dimerization interface of the MRSA-DHDPS enzyme is more extensive in buried surface area and noncovalent contacts than the equivalent interface in tetrameric DHDPS enzymes from other bacterial species
hanging-drop and sitting-drop method, solved in the native form and in complex with pyruvate at 2.3 A and 2.2 A resolution, respectively, single crystal grown in 2 M ammonium sulfate and 0.1 M Bis-Tris pH 6.5 used for data collection, processing and refinement statistics
X-ray data-collection statistics, best crystal diffracting to beyond 1.45 A resolution
by the hanging-drop vapour-diffusion method, to 2.1 A resolution. The crystal belongs to space group P42212, with unit-cell parameters a = b = 105.5, c = 62.4 A, but the R factors remain high following initial processing of the data. The data set is twinned and it is thus reprocessed in space group P2, resulting in a significant reduction in the R factors
-
purified recombinant Tm-DHDPS-DELTAArg-237, vapor diffusion method, mixing of 150 nl protein solution, containing 11.2 mg/ml protein in 20 mM Tris-HCl, pH 8.0, with 150 nl reservoir solution, containing 40% v/v PEG 300, 100 mM phosphate-citrate, buffer, pH 4.2, and 0.02% w/v sodium azide, X-ray diffraction structure determination and analysis at 1.9-2.1 A resolution
-
the X-ray crystal structure is described
crystal structure is determined at 1.92 A
purifed recombinant detagged enzyme in complex with pyruvate, hanging drop vapour diffusion method, mixing of 0.002 ml of 10 mg/ml protein in 20 mM Tris, 150 mM NaCl, pH 8.0, and 20 mM pyruvate, with 0.002 ml of reservoir solution containing 0.1 M Bis-Tris propane, pH 8.2, 0.2 M sodium bromide, and 20% w/v PEG 3350, equilibration against 1 ml of reservoir solution, 20°C, method ooptimization, X-ray diffraction structure determination and analysis at 2.2 A resolution
-
purified recombinant His-tagged enzyme, hanging drop vapor diffusion method, mixing of 0.002 ml of protein in 20 mM pyruvate and 20 mM lysine, with 0.002 ml of reservoir solution containing 0.1 M MES, pH 6.5, 6% v/v PEG 20000, X-ray diffraction structure determination and analysis at 2.40 A resolution