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Literature summary extracted from
- Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase from Lactobacillus buchneri (2017), Acta Crystallogr. Sect. D, 73, 428-437 .
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 5.1.1.21 | sequence comparisons, recombinant expression of His/ProS2-tagged wild-type and mutant enzymes | Lentilactobacillus buchneri |
Crystallization (Commentary)
| EC Number | Crystallization (Comment) | Organism |
|---|---|---|
| 5.1.1.21 | 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) | Lentilactobacillus buchneri |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 5.1.1.21 | D222A | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Lentilactobacillus buchneri |
| 5.1.1.21 | D222N | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Lentilactobacillus buchneri |
| 5.1.1.21 | Y142F | site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to the wild-type enzyme | Lentilactobacillus buchneri |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 5.1.1.21 | 4.46 | - |
L-isoleucine | recombinant mutant D222N, pH and temperature not specified in the publication | Lentilactobacillus buchneri |  |
| 5.1.1.21 | 4.58 | - |
L-isoleucine | recombinant mutant D222A, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 5 | - |
L-isoleucine | recombinant wild-type enzyme, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 13.2 | - |
D-allo-isoleucine | recombinant wild-type enzyme, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 13.8 | - |
D-allo-isoleucine | recombinant mutant D222N, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 16 | - |
D-allo-isoleucine | recombinant mutant D222A, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 16 | - |
L-isoleucine | recombinant mutant Y142F, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 35 | - |
D-allo-isoleucine | recombinant mutant Y142F, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 5.1.1.21 | L-isoleucine | Lentilactobacillus buchneri | - |
D-allo-isoleucine | - |
r | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 5.1.1.21 | Lentilactobacillus buchneri | M1GRN3 | - |
- |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 5.1.1.21 | recombinant His/ProS2-tagged wild-type and mutant enzymes, tag cleavage | Lentilactobacillus buchneri |
Reaction
| EC Number | Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|---|
| 5.1.1.21 | L-isoleucine = D-allo-isoleucine | 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 | Lentilactobacillus buchneri |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 5.1.1.21 | L-isoleucine | - |
Lentilactobacillus buchneri | D-allo-isoleucine | - |
r | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 5.1.1.21 | More | 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 | Lentilactobacillus buchneri |
| 5.1.1.21 | tetramer | the enzyme assembles as a tetramer, with each subunit being composed of N-terminal, C-terminal, and large PLP-binding domains | Lentilactobacillus buchneri |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 5.1.1.21 | BCAA racemase | UniProt | Lentilactobacillus buchneri |
| 5.1.1.21 | branched-chain amino-acid racemase | - |
Lentilactobacillus buchneri |
| 5.1.1.21 | ILEP | - |
Lentilactobacillus buchneri |
| 5.1.1.21 | isoleucine 2-epimerase | - |
Lentilactobacillus buchneri |
Turnover Number [1/s]
| EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 5.1.1.21 | 0.0238 | - |
L-isoleucine | recombinant mutant D222N, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 0.0461 | - |
D-allo-isoleucine | recombinant mutant D222N, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 1.65 | - |
L-isoleucine | recombinant mutant D222A, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 3.56 | - |
D-allo-isoleucine | recombinant mutant D222A, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 279 | - |
L-isoleucine | recombinant mutant Y142F, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 434 | - |
D-allo-isoleucine | recombinant mutant Y142F, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 502 | - |
L-isoleucine | recombinant wild-type enzyme, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 939 | - |
D-allo-isoleucine | recombinant wild-type enzyme, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
Cofactor
| EC Number | Cofactor | Comment | Organism | Structure |
|---|---|---|---|---|
| 5.1.1.21 | pyridoxal 5'-phosphate | 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 | Lentilactobacillus buchneri | |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 5.1.1.21 | evolution | the enzyme is a fold-type I racemase | Lentilactobacillus buchneri |
| 5.1.1.21 | additional information | 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 | Lentilactobacillus buchneri |
kcat/KM [mM/s]
| EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 5.1.1.21 | 0.00334 | - |
D-allo-isoleucine | recombinant mutant D222N, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 0.00534 | - |
L-isoleucine | recombinant mutant D222N, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 0.223 | - |
D-allo-isoleucine | recombinant mutant D222A, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 0.361 | - |
L-isoleucine | recombinant mutant D222A, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 12.4 | - |
D-allo-isoleucine | recombinant mutant Y142F, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 17.5 | - |
L-isoleucine | recombinant mutant Y142F, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 71.4 | - |
D-allo-isoleucine | recombinant wild-type enzyme, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
| 5.1.1.21 | 101 | - |
L-isoleucine | recombinant wild-type enzyme, pH and temperature not specified in the publication | Lentilactobacillus buchneri | |
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