EC Number | Application | Comment | Organism |
---|---|---|---|
3.5.1.100 | synthesis | R-amidase is the first enzyme useful for the enzymatic optical resolution of racemic piperazine-2-tert-butylcarboxamide carried out under mild conditions. Enantiomerically pure piperazine-2-carboxylic acid and its tert-butylcarboxamide derivative are important chiral building blocks for some pharmacologically active compounds such as N-methyl-D-aspartate antagonist for glutamate receptor, cardioprotective nucleoside transport blocker, and HIV protease inhibitor | Pseudomonas sp. |
EC Number | Cloned (Comment) | Organism |
---|---|---|
3.5.1.100 | expression in Escherichia coli | Pseudomonas sp. |
EC Number | Inhibitors | Comment | Organism | Structure |
---|---|---|---|---|
3.5.1.100 | AgNO3 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | CdCl2 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | CoCl2 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | CuCl2 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | CuSO4 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | Fe(NH4)2(SO4)2 | 1 mM, incubation at 30°C for 10 min, 67% inhibition | Pseudomonas sp. | |
3.5.1.100 | FeCl3 | 1 mM, incubation at 30°C for 10 min, 78% inhibition | Pseudomonas sp. | |
3.5.1.100 | HgCl2 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | MnCl2 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | MnSO4 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | additional information | chelating reagents, e.g. o-phenanthroline, 8-hydroxyquinoline, ethylenediaminetetraacetic acid and 2,2'-dipyridyl have no significant effect on the enzyme. Carbonyl reagents such as hydroxylamine, phenylhydrazine, hydrazine, DL-penicillamine and D-cycloserine are not inhibitory toward the enzyme | Pseudomonas sp. | |
3.5.1.100 | N-ethylmaleimide | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | NiCl2 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | p-chloromercuribenzoate | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | PbCl2 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | ZnCl2 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. | |
3.5.1.100 | ZnSO4 | 1 mM, incubation at 30°C for 10 min, complete inhibition | Pseudomonas sp. |
EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|---|
3.5.1.100 | 29500 | - |
1 * 29500, SDS-PAGE | Pseudomonas sp. |
3.5.1.100 | 30128 | - |
1 * 30128, calculated from sequence | Pseudomonas sp. |
3.5.1.100 | 36000 | - |
gel filtration | Pseudomonas sp. |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
3.5.1.100 | Pseudomonas sp. | Q75SP7 | - |
- |
3.5.1.100 | Pseudomonas sp. MCI3434 | Q75SP7 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
3.5.1.100 | - |
Pseudomonas sp. |
EC Number | Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|---|
3.5.1.100 | 0.0242 | - |
R-amidase from Pseudomonas sp. MCI3434 | Pseudomonas sp. |
3.5.1.100 | 4.59 | - |
RamA from Escherichia coli JM109 harboring pRTB1EX | Pseudomonas sp. |
EC Number | Storage Stability | Organism |
---|---|---|
3.5.1.100 | -20°C, stable for more than 2 months in the buffer containing 50% glycerol | Pseudomonas sp. |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
3.5.1.100 | (R)-piperazine-2-carboxamide + H2O | hydrolysis with strict R-stereoselectivity | Pseudomonas sp. | (R)-piperazine-2-carboxylic acid + NH3 | - |
? | |
3.5.1.100 | (R)-piperazine-2-carboxamide + H2O | hydrolysis with strict R-stereoselectivity | Pseudomonas sp. MCI3434 | (R)-piperazine-2-carboxylic acid + NH3 | - |
? | |
3.5.1.100 | (R)-piperazine-2-tert-butylcarboxamide + H2O | hydrolysis with strict R-stereoselectivity, 9% of the activity with (R)-piperazine-2-carboxamide | Pseudomonas sp. | (R)-piperazine-2-carboxylate + tert-butylamine | - |
? | |
3.5.1.100 | (R)-piperazine-2-tert-butylcarboxamide + H2O | hydrolysis with strict R-stereoselectivity, 9% of the activity with (R)-piperazine-2-carboxamide | Pseudomonas sp. MCI3434 | (R)-piperazine-2-carboxylate + tert-butylamine | - |
? | |
3.5.1.100 | (R)-piperidine-3-carboxamide + H2O | 68.9% of the activity with (R)-piperazine-2-carboxamide | Pseudomonas sp. | (R)-piperidine-3-carboxylic acid + NH3 | - |
? | |
3.5.1.100 | (R)-piperidine-3-carboxamide + H2O | 68.9% of the activity with (R)-piperazine-2-carboxamide | Pseudomonas sp. MCI3434 | (R)-piperidine-3-carboxylic acid + NH3 | - |
? | |
3.5.1.100 | beta-alaninamide + H2O | 108% of the activity with (R)-piperazine-2-carboxamide | Pseudomonas sp. | beta-alanine + NH3 | - |
? | |
3.5.1.100 | beta-alaninamide + H2O | 108% of the activity with (R)-piperazine-2-carboxamide | Pseudomonas sp. MCI3434 | beta-alanine + NH3 | - |
? | |
3.5.1.100 | D-glutamine amide + H2O | no formation of glutamine, 27% of the activity with (R)-piperazine-2-carboxamide | Pseudomonas sp. | D-glutamic acid + NH3 | - |
? | |
3.5.1.100 | L-glutamine amide + H2O | no formation of glutamine, 0.35% of the activity with (R)-piperazine-2-carboxamide | Pseudomonas sp. | L-glutamic acid + NH3 | - |
? | |
3.5.1.100 | additional information | RamA has hydrolyzing activity toward the carboxamide compounds, in which an amino or imino group is connected to beta- or gamma-carbon, such as beta-alanine amide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutamine amide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme does not act on the other amide substrates for the aliphatic amidase: D-alanine amide, D-valine amide, D-leucine amide, D-isoleucine amide, D-proline amide, D-phenylalanine amide, D-tryptophan amide, D-methionine amide, D-serine amide, D-threoninamide, D-tyrosine amide, D-aspartic acid amide, D-glutamic acid amide, D-lysine amide, D-arginine amide, D-histidine amide, L-alanine amide, L-valine amide, L-leucine amide, L-isoleucine amide, L-proline amide, L-phenylalanine amide, L-tryptophanamide, L-methioninamide, L-serine amide, L-threonine amide, L-tyrosine amide, L-asparagine amide, L-aspartic acid amide, L-glutamic acid amide, L-lysine amide, L-arginine amide, L-histidine amide, glycine amide and (R,S)-piperidine-2-carboxamide. Carboxamides of the side chains in D-asparagine, D-glutamine, L-asparagine and L-glutamine are not hydrolyzed by the enzyme. The enzyme does not show peptidase activity toward beta-alanyl-L-alanine, beta-alanylglycine, beta-alanyl-L-histidine, glycylglycine, glycylglycylglycine, L-alanylglycine, D-alanylglycine, D-alanylglycylglycine, DL-alanyl-DL-asparagine, DL-alanyl-DL-isoleucine, DL-alanyl-DL-leucine, DL-alanyl-DL-methionine, DL-alanyl-DL-phenylalanine, DL-alanyl-DL-serine, DL-alanyl-DL-valine and L-aspartyl-D-alanine. The enzyme could not hydrolyze the following aliphatic amides, aromatic amides and nitriles: acetamide, propionamide, n-butyramide, isobutyramide, n-valeramide, n-capronamide, crotonamide, methacrylamide, cyclohexanecarboxamide, benzamide, o-aminobenzamide, m-aminobenzamide, p-aminobenzamide, p-toluamide, p-chlorobenzamide, p-nitrobenzamide, 2-picolinamide, nicotinamide, pyridine-4-carboxamide, pyrazinamide,2-thiophenecarboxamide, phenylacetamide, indole-3-acetamide, acetonitrile, propionitrile, 3-hydroxypropionitrile, n-capronitrile, methacrylonitrile, crotononitrile, glutaronitrile, 2,4-dicyanobut-1-ene, beta-phenylpropionitrile, cinnamonitrile, 2-cyanopiperidine, 2-cyanopiperazine, phenylacetonitrile, 4-methoxyphenylacetonitrile, alpha-methylbenzyl cyanide, 2-pyridineacetonitrile, 3-pyridineacetonitrile, thiophene-2-acetonitrile, beta-indoleacetonitrile, diphenylacetonitrile, 4-chlorobenzyl cyanide, benzonitrile, 4-chlorobenzonitrile, 4-nitrobenzonitrile, p-tolunitrile, anisonitrile, 2-cyanophenol, 2-cyanopyridine, 3-cyanopyridine, 4-cyanopyridine, pyrazinecarbonitrile, 3-cyanoindole, a-naphthonitrile, 2-thiophenecarbonitrile, terephthalonitrile and isophthalonitrile | Pseudomonas sp. | ? | - |
? | |
3.5.1.100 | additional information | RamA has hydrolyzing activity toward the carboxamide compounds, in which an amino or imino group is connected to beta- or gamma-carbon, such as beta-alanine amide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutamine amide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme does not act on the other amide substrates for the aliphatic amidase: D-alanine amide, D-valine amide, D-leucine amide, D-isoleucine amide, D-proline amide, D-phenylalanine amide, D-tryptophan amide, D-methionine amide, D-serine amide, D-threoninamide, D-tyrosine amide, D-aspartic acid amide, D-glutamic acid amide, D-lysine amide, D-arginine amide, D-histidine amide, L-alanine amide, L-valine amide, L-leucine amide, L-isoleucine amide, L-proline amide, L-phenylalanine amide, L-tryptophanamide, L-methioninamide, L-serine amide, L-threonine amide, L-tyrosine amide, L-asparagine amide, L-aspartic acid amide, L-glutamic acid amide, L-lysine amide, L-arginine amide, L-histidine amide, glycine amide and (R,S)-piperidine-2-carboxamide. Carboxamides of the side chains in D-asparagine, D-glutamine, L-asparagine and L-glutamine are not hydrolyzed by the enzyme. The enzyme does not show peptidase activity toward beta-alanyl-L-alanine, beta-alanylglycine, beta-alanyl-L-histidine, glycylglycine, glycylglycylglycine, L-alanylglycine, D-alanylglycine, D-alanylglycylglycine, DL-alanyl-DL-asparagine, DL-alanyl-DL-isoleucine, DL-alanyl-DL-leucine, DL-alanyl-DL-methionine, DL-alanyl-DL-phenylalanine, DL-alanyl-DL-serine, DL-alanyl-DL-valine and L-aspartyl-D-alanine. The enzyme could not hydrolyze the following aliphatic amides, aromatic amides and nitriles: acetamide, propionamide, n-butyramide, isobutyramide, n-valeramide, n-capronamide, crotonamide, methacrylamide, cyclohexanecarboxamide, benzamide, o-aminobenzamide, m-aminobenzamide, p-aminobenzamide, p-toluamide, p-chlorobenzamide, p-nitrobenzamide, 2-picolinamide, nicotinamide, pyridine-4-carboxamide, pyrazinamide,2-thiophenecarboxamide, phenylacetamide, indole-3-acetamide, acetonitrile, propionitrile, 3-hydroxypropionitrile, n-capronitrile, methacrylonitrile, crotononitrile, glutaronitrile, 2,4-dicyanobut-1-ene, beta-phenylpropionitrile, cinnamonitrile, 2-cyanopiperidine, 2-cyanopiperazine, phenylacetonitrile, 4-methoxyphenylacetonitrile, alpha-methylbenzyl cyanide, 2-pyridineacetonitrile, 3-pyridineacetonitrile, thiophene-2-acetonitrile, beta-indoleacetonitrile, diphenylacetonitrile, 4-chlorobenzyl cyanide, benzonitrile, 4-chlorobenzonitrile, 4-nitrobenzonitrile, p-tolunitrile, anisonitrile, 2-cyanophenol, 2-cyanopyridine, 3-cyanopyridine, 4-cyanopyridine, pyrazinecarbonitrile, 3-cyanoindole, a-naphthonitrile, 2-thiophenecarbonitrile, terephthalonitrile and isophthalonitrile | Pseudomonas sp. MCI3434 | ? | - |
? | |
3.5.1.100 | piperidine-4-carboxamide + H2O | 0.23% of the activity with (R)-piperazine-2-carboxamide | Pseudomonas sp. | piperidine-4-carboxylic acid + NH3 | - |
? |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
3.5.1.100 | monomer | 1 * 29500, SDS-PAGE | Pseudomonas sp. |
3.5.1.100 | monomer | 1 * 30128, calculated from sequence | Pseudomonas sp. |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
3.5.1.100 | R-amidase | - |
Pseudomonas sp. |
3.5.1.100 | R-stereoselective amidase | - |
Pseudomonas sp. |
3.5.1.100 | RamA | - |
Pseudomonas sp. |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
3.5.1.100 | 45 | - |
- |
Pseudomonas sp. |
EC Number | Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|---|
3.5.1.100 | 35 | - |
10 min, stable | Pseudomonas sp. |
3.5.1.100 | 40 | - |
10 min, stable | Pseudomonas sp. |
3.5.1.100 | 45 | - |
10 min, 13% loss of activity | Pseudomonas sp. |
3.5.1.100 | 50 | - |
10 min, 97% loss of activity | Pseudomonas sp. |
3.5.1.100 | 55 | - |
10 min, complete loss of activity | Pseudomonas sp. |
EC Number | pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|---|
3.5.1.100 | 6 | 9 | 30°C, 10 min, most stable in pH-range 6.0-9.0 | Pseudomonas sp. |