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5.3.3.1: steroid DELTA-isomerase

This is an abbreviated version!
For detailed information about steroid DELTA-isomerase, go to the full flat file.

Word Map on EC 5.3.3.1

Reaction

a 3-oxo-DELTA5-steroid
=
a 3-oxo-DELTA4-steroid

Synonyms

3-Keto-DELTA5-steroid isomerase, 3-Ketosteroid DELTA5-->DELTA4-isomerase, 3-ketosteroid isomerase, 3-Oxo steroid DELTA4-DELTA5-isomerase, 3-Oxo-delta5 steroid isomerase, 3-oxo-DELTA5-steroid isomerase, 3-Oxosteroid DELTA4-DELTA5-isomerase, 3-Oxosteroid DELTA5-DELTA4-isomerase, 3-Oxosteroid isomerase, 3beta-HSD, 3beta-HSD/isomerase, 3beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase, 3beta-hydroxysteroid dehydrogenase/DELTA5-DELTA4 isomerase, 3beta-hydroxysteroid dehydrogenase/isomerase, 3beta-hydroxysteroid dehydrogenase/isomerase type 1, 3beta-hydroxysteroid dehydrogenase/isomerase type 2, 3KSI, 5-Ene-4-ene isomerase, 5-Pregnene-3,20-dione isomerase, delta 5-3-ketosteroid isomerase, DELTA-3-ketosteroid isomerase, Delta-5-3-ketosteroid isomerase, DELTA5(or DELTA4)-3-keto steroid isomerase, DELTA5-3-keto steroid isomerase, DELTA5-3-ketosteroid isomerase, DELTA5-3-oxosteroid isomerase, DELTA5-ketosteroid isomerase, DELTA5-steroid isomerase, glutathione transferase A3-3, GST A3-3, Hydroxysteroid isomerase, Isomerase, steroid DELTA, ketosteroid isomerase, KSI, More, Steroid 5-->4-isomerase, Steroid isomerase, TI, TI-WT, type I 3beta-hydroxysteroid dehydrogenase/isomerase

ECTree

     5 Isomerases
         5.3 Intramolecular oxidoreductases
             5.3.3 Transposing C=C bonds
                5.3.3.1 steroid DELTA-isomerase

Crystallization

Crystallization on EC 5.3.3.1 - steroid DELTA-isomerase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
1.2-1.5 A resolution X-ray crystallography, 1H and 19F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements of the active site. Packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 A or less. This constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. Structural features of the oxyanion hole suggest that hydrogen bond formation to the reacting substrate is geometrically optimal in the transition state but not in the ground state. During steroid isomerization, the hybridization of the substrate oxygen changes from a planar sp2 carbonyl to a tetrahedral sp3 dienolate, altering the spatial distribution of its lone pair electrons. This reorientation of atomic orbitals about the substrate oxygen alters its geometric preference for accepting hydrogen bonds
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crystals are grown at 12°C from1.3 M ammonium sulfate, 3-5% poly(ethylene glycol)400 and 0.1 M HEPES, pH 7.5 in hanging drops. The crystal structure at 2.3 resolution reveals that the active site environment of the Comamonas testosteroni enzyme is nearly identical to that of Pseudomonas putida enzyme
crystals of mutant enzyme F116W are grown by hanging-drop method
study on backbone dynamics in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate. Mutation Y14F induces a substantial decrease in the order parameters in free enzyme, indicating that the backbone structures become significantly mobile by mutation, while the chemical shift analysis indicates that the structural perturbations are more profound than those of wild-type upon 19-nortestosterone hemisuccinate binding. In the 19-nortestosterone hemisuccinate complexed mutant, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remain flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in the mutant enzyme become even greater than those of wild-type
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The 2.26 A crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals that both the Tyr14 OH and the Asp99 COOH provide direct hydrogen bonds to the oxyanion of equilenin
GST A3-3 in complex with DELTA5-androstene-3,17-dione, using 100 mM Tris-HCl pH 7.8, 18% (v/v) PEG 4000, and 2 mM dithiothreitol
hanging-drop vapor diffusion method. The inability to crystallize the detergent-solubilized, wild-type microsomal enzyme is overcome by engineering a cytosolic form of this protein. Modified enzyme in which the membrane-spanning domain, residues 283-310 of the enzyme protein is deleted in the cDNA is expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells
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identification of potentially critical residues M187 and S124 by docking of trilostane or 4alpha,5alpha-epoxy-testosterone into the active site
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1.2-1.5 A resolution X-ray crystallography, 1H and 19F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements of the active site. Packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 A or less. This constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. Structural features of the oxyanion hole suggest that hydrogen bond formation to the reacting substrate is geometrically optimal in the transition state but not in the ground state. During steroid isomerization, the hybridization of the substrate oxygen changes from a planar sp2 carbonyl to a tetrahedral sp3 dienolate, altering the spatial distribution of its lone pair electrons. This reorientation of atomic orbitals about the substrate oxygen alters its geometric preference for accepting hydrogen bonds
crystal structure of mutant enzyme F82A is determined to 2.1 A resolution. Crystals are grown in a solution containing 1.0 M sodium acetate and 0.1 M ammonioum acetate, pH 4.6, by the hanging drop method of vapor diffusion at 22 C. The crystals belong to the space group c2221 with unit cell dimensions of a = 36.24 A, b = 96.13 A and c = 74.30 A
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crystal structure of the enzyme in complex with equilenin, an analogue of the reaction intermediate at 1.9 and 2.5 A resolution
crystal structure of the R72A mutant enzyme determined at 2.5 A resolution belongs to the space group C2221 with cell dimensions of a = 36.37 A, b = 74.44 A and c = 96.06 A. Crystals are grown from a solution containing 2.0 M ammonioum acetate and 0.1 M sodium acetate at pH 4.6 by hanging drop vapor-diffusion method at 22°C
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crystals of Y30F, Y55F, and Y30F/Y55F are grown in the solution containing 1.0 M sodium acetate and 0.1 M ammonium acetate, pH 4.6 by hanging drop method of vapor diffusion at 22°C. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30
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enzyme mutant D40N bound to phenolate, X-ray diffraction structure determination and analysis at 1.25 A resolution
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hanging drop vapor diffusion method. Crystal structures of Y14F and Y14F/Y30F/Y55F are determined at 1.8 and 2.0 A resolution, respectively
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mutant D103N/D40N bound to inhibitor equilenin, hanging drop vapor diffusion method, 0.002 ml of 25 mg/ml protein with equilenin in a molar ratio of 1:1.2 in 40 mM potassium phosphate, pH 7.2, are mixed with 0.002 ml of reservoir solution conraining 1.4 M ammonium sulfate, and 6.5% v/v 2-propanol, pH 7.0, room temperature, 1 week, X-ray diffraction structure determination and analysis at 1.1 A resolution
mutant enzyme D99E/D38N complexed with equilenin, an intermediate analogue, crystals of the complexes are grown from 1.1 M ammonium acetate and 0.1 M sodium acetate, pH 4.6, by the hanging drop vapor diffusion method at 22°C. The resulting crystals have C2 space group symmetry with unit cell dimensions of a = 89.04 A, b = 72.42 A, c = 51.24 A and beta = 90.9°
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mutant enzyme Y30F/Y55F/Y115F/D38N KSI complexed with equilenin, hanging drop vapor diffusion method, using 0.1 M sodium acetate, pH 4.5, 0.6 M ammonium acetate, and 30% PEG 4000
mutant W92A, in complex with d-equilenin, decrease in conformational stability results from destabilization of surface hydrophobic layer
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mutant Y14F/D99L, increase in hydrophobic interaction while disrupting the hydrogen bond network, mutants Y30F/D99L and Y55F/D99L, disruption of hydrogen bond network
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