A pyridoxal phosphate protein. The enzyme, characterized from the bacterium Lactobacillus buchneri, specifically catalyses racemization of nonpolar amino acids at the C-2 position.
The taxonomic range for the selected organisms is: Lentilactobacillus buchneri The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
L-isoleucine epimerization proceeds through abstraction of the alpha-hydrogen of the substrate by Lys280, while Asp222 serves as the catalytic residue adding an alpha-hydrogen to the quinonoid intermediate to form D-allo-isoleucine, structure-function relationship
the racemization reaction by the fold-type I racemases may generally occur thanks to a revised two-base mechanism. Conformational changes provoked by pyridoxal 5'-phosphate binding suggesting an induced fit of the active site entrance door necessary to accommodate pyridoxal 5'-phosphate and substrate molecules
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SYSTEMATIC NAME
IUBMB Comments
isoleucine 2-epimerase
A pyridoxal phosphate protein. The enzyme, characterized from the bacterium Lactobacillus buchneri, specifically catalyses racemization of nonpolar amino acids at the C-2 position.
the enzyme specifically catalyzes racemization of nonpolar amino acids at the C-2 position. No activity toward the L- and D-forms of Asp, Glu, Lys, Arg, His, Orn, Thr, Asn, Gln, Trp, and tert-Leu
the enzyme specifically catalyzes racemization of nonpolar amino acids at the C-2 position. No activity toward the L- and D-forms of Asp, Glu, Lys, Arg, His, Orn, Thr, Asn, Gln, Trp, and tert-Leu
the enzyme catalyzes the pyridoxal 5'-phosphate (PLP)-dependent racemization and epimerization of a broad spectrum of nonpolar amino acids from L- to D-form and vice versa, in particular isoleucine
the enzyme catalyzes the pyridoxal 5'-phosphate (PLP)-dependent racemization and epimerization of a broad spectrum of nonpolar amino acids from L- to D-form and vice versa, in particular isoleucine
PLP, a marked structural change occurs around the active site upon binding of pyridoxal 5'-phosphate that provides a solvent-inaccessible environment for the enzymatic reaction
almost fully inactivated by the first dialysis against buffer containing 50 mM hydroxylamine, after which about 75% of the activity is recovered by a subsequent dialysis against buffer containing 1 mM pyridoxal 5'-phosphate
identification of the active site residues responsible for its nonpolar amino acid recognition and reactivity specificity from structure comparisons with the alpha-amino-epsilon-caprolactam racemase (EC 5.1.1.15) from Achromobacter obae and the cystathionine beta-lyase (EC 4.4.1.8) from Escherichia coli (PDB IDs 2ZUK and 3DXW), active site structure, overview
identification of the active site residues responsible for its nonpolar amino acid recognition and reactivity specificity from structure comparisons with the alpha-amino-epsilon-caprolactam racemase (EC 5.1.1.15) from Achromobacter obae and the cystathionine beta-lyase (EC 4.4.1.8) from Escherichia coli (PDB IDs 2ZUK and 3DXW), active site structure, overview
the enzyme is a fold-type I racemase, similar to the alpha-amino-epsilon-caprolactam racemase (EC 5.1.1.15). The active-site cavity in the apoenzyme structure is much more solvent-accessible than that in the pyridoxal 5'-phosphate-bound structure. A marked structural change occurs around the active site upon binding of pyridoxal 5'-phosphate that provides a solvent-inaccessible environment for the enzymatic reaction. Detailed enzyme structure analysis and structure comparisons, active site and substrate/ligand-binding structre, structure-function relationship, overview
the enzyme is a fold-type I racemase, similar to the alpha-amino-epsilon-caprolactam racemase (EC 5.1.1.15). The active-site cavity in the apoenzyme structure is much more solvent-accessible than that in the pyridoxal 5'-phosphate-bound structure. A marked structural change occurs around the active site upon binding of pyridoxal 5'-phosphate that provides a solvent-inaccessible environment for the enzymatic reaction. Detailed enzyme structure analysis and structure comparisons, active site and substrate/ligand-binding structre, structure-function relationship, overview
detailed enzyme structure analysis and structure comparisons, analysis of the structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile), overview
detailed enzyme structure analysis and structure comparisons, analysis of the structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile), overview
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant detagged enzyme in apoform, in complex with pyridoxal 5'-phosphate, in complex with N-(5'-phosphopyridoxyl)-L-isoleucine, and in complex with N-(5'-phosphopyridoxyl)-D-allo-isoleucine. By sitting drop vapour diffusion method, for crystals of PLP-bound enzyme, mixing of 0.002 ml enzyme solution containing 0.05 mM PLP 33% 2-propanol, 0.2 M magnesium acetate, 0.1 M cacodylate buffer pH 6.5, for crystals of the apoenzyme mixing of 0.001 ml enzyme solution containing 2 mM D-tert-Leu and 0.1 mM PLP with 0.001 ml of 0.5 M NaCl, 10 mM MgCl2, 10mM hexadecyltrimethylammonium bromide, for crystals of the PLP-L-Ile-bound enzyme, mixing of 0.001 ml protein solution with 0.001 ml of PLP-L-Ile, 20% PEG 3000, 0.2 M NaCl, 0.1 M HEPES, pH 7.5, and for crystals of the PLP-D-allo-Ile-bound enzyme are grown in sitting drops composed of 0.001 ml enzyme solution containing 1 mM PLP-D-allo-Ile mixed with an equal volume of 50 mM cadmium sulfate hydrate, 0.76 M sodium acetate trihydrate, and 0.1 M HEPES, pH 7.5. In all cases, drops are equilibrated against 0.1 ml mother liquor at 20°C, X-ray diffraction structure determination and analysis at resolutions of 2.77 A, 1.94 A, 2.65 A, and 2.12 A, respectively, molecular replacement using with the tetrameric structure of GABA-AT from Sulfolobus tokodaii (PDB ID 2eo5)
purified recombinant His-tagged enzyme alone or in complex with pyridoxal 5'-phosphate, hanging drop vapour diffusion technique, mixing of 0.002 ml of 2-8 mg/ml protein in 50 mM Na-phosphate, pH 7.2, and 0.1 mM 2-mercaptoethanol, with 0.002 ml of reservoir solution containing 12-18% PEG 3350 and 100 mM lithium citrate, 20°C, several weeks, for complex crystals soaking in pyridoxal 5'-phosphate containing mother liquor, X-ray diffraction structure determination and analysis at 2.6 A and 2.15 A resolution, respectively
Structural insights into the substrate recognition and reaction specificity of the PLP-dependent fold-type I isoleucine 2-epimerase from Lactobacillus buchneri