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Literature summary for 4.1.2.47 extracted from
- Identification of potential active-site residues in the hydroxynitrile lyase from Manihot esculenta by site-directed mutagenesis (1996), J. Biol. Chem., 271, 25830-25834.
Application
| Application | Comment | Organism |
|---|---|---|
| synthesis | synthesis of optically active cyanohydrins that are interesting intermediates for the synthesis of alpha-hydroxy acids, alpha-hydroxy ketones, or beta-ethanolamines, all of which are important building blocks in organic synthesis | Manihot esculenta |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression in Escherichia coli | Manihot esculenta |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D208A | Km-value for 2-hydroxy-2-methylpropanenitrile increases from 101 mM for wild-type enzyme to over 200 mM for the mutant enzyme | Manihot esculenta |
| H10A | limited decrease in activity | Manihot esculenta |
| H112A | limited decrease in activity | Manihot esculenta |
| H236A | mutant enzyme is unable to catalyze the decomposition of 2-hydroxy-2-methylpropanenitrile | Manihot esculenta |
| H5A | limited decrease in activity | Manihot esculenta |
| S80A | mutant enzyme is completely inactive in the 2-hydroxy-2-methylpropanenitrile cleaving assay. No differences to wild type MeHNL according to oligomeric structure, molecular weight, and behavior in the standard purification procedure | Manihot esculenta |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride | - |
Manihot esculenta |  |
| diisopropyl fluorophosphate | both natural MeHNL as well as recombinant MeHNL are almost completely inhibited by diisopropyl fluorophosphate at 1 mM | Manihot esculenta | |
| additional information | leupeptin and pepstatin A do not affect the MeHNL-catalyzed 2-hydroxy-2-methylpropanenitrile cleavage at pH 5.4 | Manihot esculenta | |
| phenylmethylsulfonyl fluoride | - |
Manihot esculenta | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 95 | - |
2-hydroxy-2-methylpropanenitrile | mutant enzyme H112A | Manihot esculenta | |
| 101 | - |
2-hydroxy-2-methylpropanenitrile | recombinant enzyme | Manihot esculenta | |
| 120 | - |
2-hydroxy-2-methylpropanenitrile | wild-type enzyme | Manihot esculenta | |
| 120 | - |
2-hydroxy-2-methylpropanenitrile | mutant enzyme D130A | Manihot esculenta | |
| 120 | - |
2-hydroxy-2-methylpropanenitrile | mutant enzyme D95A | Manihot esculenta | |
| 125 | - |
2-hydroxy-2-methylpropanenitrile | mutant enzyme H5A | Manihot esculenta | |
| 130 | - |
2-hydroxy-2-methylpropanenitrile | mutant enzyme H10A | Manihot esculenta | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2-hydroxy-2-methylpropanenitrile | Manihot esculenta | i.e. acetone cyanohydrin, catalyzes the decomposition of the achiral alpha-hydroxynitrile 2-hydroxy-2-methylpropanenitrile into HCN and acetone during cyanogenesis of damaged plants | cyanide + acetone | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Manihot esculenta | P52705 | Crantz | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant enzyme | Manihot esculenta |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| an aliphatic (S)-hydroxynitrile = cyanide + an aliphatic aldehyde or ketone | proposed reaction mechanism: in free enzyme, the hydroxyl group of Ser80 is hydrogen-bonded to the imidazole nitrogen of His236, which in turn is stabilized by a hydrogen bond with Asp208. If the enzyme encounters a substrate molecule, the proton of Ser80 is rapidly transferred to an imidazole nitrogen of His236. The resulting oxyanion of Ser80 functions as a strong base in a nucleophilic attack of the substrate hydroxyl proton, leading to a negatively charged oxyanion on the substrate. This oxyanion could be stabilized by an oxygen hole formed by amide nitrogen of the backbone of Ser80 and Gly78. Both residues belong to the consensus motif, which is typical for nucleophils located in a catalytic triad. The stabilized oxyanion could further increase the local positive charge on the alpha-C atom of the cyanohydrin, allowing the cyanide group to leave the molecule | Manihot esculenta |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| leaf | - |
Manihot esculenta | - |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 86 | - |
recombinant enzyme | Manihot esculenta |
| 92 | - |
wild-type enzyme from leaves | Manihot esculenta |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2-hydroxy-2-methylpropanenitrile | i.e. acetone cyanohydrin | Manihot esculenta | cyanide + acetone | - |
? | |
| 2-hydroxy-2-methylpropanenitrile | i.e. acetone cyanohydrin, catalyzes the decomposition of the achiral alpha-hydroxynitrile 2-hydroxy-2-methylpropanenitrile into HCN and acetone during cyanogenesis of damaged plants | Manihot esculenta | cyanide + acetone | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| MeHNL | - |
Manihot esculenta |
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