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Literature summary extracted from

  • Komeda, H.; Harada, H.; Washika, S.; Sakamoto, T.; Ueda, M.; Asano, Y.
    A novel R-stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-tert-butylcarboxamide (2004), Eur. J. Biochem., 271, 1580-1590.
    View publication on PubMed

Application

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.

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.100 expression in Escherichia coli Pseudomonas sp.

Inhibitors

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.

Molecular Weight [Da]

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.

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.100 Pseudomonas sp. Q75SP7
-
-
3.5.1.100 Pseudomonas sp. MCI3434 Q75SP7
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.100
-
 Pseudomonas sp.

Specific Activity [micromol/min/mg]

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.

Storage Stability

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.

Substrates and Products (Substrate)

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
-
 ?

Subunits

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.

Synonyms

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.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.100 45
-
-
 Pseudomonas sp.

Temperature Stability [°C]

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.

pH Stability

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.